JP5508654B2 - Method for designing positionable arrays for detection of nucleic acid sequence differences using ligase detection reactions - Google Patents

Description

  The present invention was developed using government funds from National Institutes of Health grant numbers GM-41337-06, GM-43552-05, GM-42722-07 and GM-51628-02. The US government may have certain rights.

The present invention relates to a plurality of capture oligonucleotides for use on a support in which a plurality of capture oligonucleotide probes have a narrow range of melting temperatures and to which complementary oligonucleotides hybridize with little mismatch. The present invention relates to a method for designing a probe. Another aspect of the invention is a support having a plurality of oligonucleotide probes immobilized on a support, a support for detecting single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences And a kit for such detection comprising a support on which oligonucleotides are immobilized.

Background of the Invention
Detection of sequence differences Large-scale multiplex analysis of highly polymorphic loci can be found in paternity testing and forensic science (Reynolds et al., Anal. Chem., 63: 2-15 (1991)), donors and recipes in organ transplantation. Combinations of ents (Buyse et al., Tissue Antigens, 41: 1-14 (1993) and Gyllensten et al., PCR Meth. Appl. 1: 91-98 (1991)), diagnosis of genetic disease, prognosis and prenatal counseling (Chamberlain et al. Nucleic Acids Res., 16: 11141-11156 (1988) and LCTsui, Human Mutat., 1: 197-203 (1992)), and oncogenic mutation studies (Hollstein et al., Science, 253: 49-53 (1991). )) Etc. to help with identification of individuals. In addition, the cost effectiveness of infectious disease diagnosis by nucleic acid analysis varies greatly depending on the multiplicity of panel tests. Many of the above applications are based on the identification of single base differences at various loci that may be spatially close.

  A variety of DNA hybridization methods can be utilized to detect the presence of one or more polynucleotide sequences in a sample containing a region of multiple sequences. In a simple method based on fragment capture and labeling, a fragment containing a specific sequence is captured by hybridization to an immobilized probe. The capture fragment is labeled by hybridization with another probe containing a reporter moiety for detection.

  Another widely used method is the Southern blot method. In this method, a mixture of DNA fragments in a sample is fractionated by gel electrophoresis and then fixed to a nitrocellulose filter. By reacting the filter with one or more labeled probes under hybridization conditions, the presence of a band containing the probe sequence can be identified. This method is particularly useful for the identification of fragments (including any probe sequences) in DNA digests by restriction enzymes and the analysis of restriction fragment length polymorphisms ("RFLP").

  Other methods for detecting the presence of any sequence or group of sequences contained in a polynucleotide sample include the polymerase chain reaction (US Pat. No. 4,683,202 by Mullis et al., And the paper by RKSaiki et al. (Science). 230: 1350 (1985))) and selective amplification of sequences (or sequences). In this method, a primer that is complementary to the opposite end of a specific sequence (or group of sequences) is used to drive successive replications initiated by the primer in a heating cycle. The amplified sequence can be easily identified by various methods. The method is particularly useful in detecting low copy sequences contained in a polynucleotide sample, for example, detection of pathogen sequences in a body fluid sample.

  More recently, methods for identifying known target sequences by probe ligation methods include US Pat. No. 4,883,750 by NMWhiteley et al., DYWu et al. (Genomics 4: 560 (1989)), This is described in a paper by U. Landegren et al. (Science 241: 1077 (1988)) and a paper by E. Winn-Deen et al. (Clin. Chem. 37: 1522 (1991)). In a method known as oligonucleotide ligation assay (OLA), a hybrid is formed between two probes or probe elements spanning the target region of interest and the target region. When the probe element is paired with an adjacent target base at the facing end of the probe element (forms a base pair), the two elements can be ligated, eg, treated with ligase. An assay is then performed on the linked probe elements to determine the presence of the target sequence.

  In a variation of this approach, the linking probe element serves as a template for a complementary probe element pair. By continuing the cycle of denaturation, hybridization and ligation in the presence of two complementary probe element pairs, the target sequence is amplified exponentially (ie, exponentially) to detect and detect trace amounts of target sequence. / Or amplified. This method is called ligase chain reaction (“LCR”) (F. Barany “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase”) (Proc. Acad. Sci. USA 88: 189-93 (1991) and F. Barany, “The Ligase Chain Reaction (LCR) in a PCR World” (PCR Methods and Applications) 1: 5-16 (1991)).

  US Pat. No. 5,470,705 to Grossman et al. Which has a label for detection and is capable of hybridizing and linking the sequence-specific probe and target with a significant [charge] / [frictional resistance associated with translation] ratio. Discloses another method for multiplex detection of nucleic acid sequence differences. This method is described in a paper by Grossman et al. ("High-density Multiplex Detection of Nucleic Acid Sequences: Oligonucleotide Ligation Assay and Sequence-coded Separation" Nucleic Acids Res., 22 (21): 4527-34 (1994)). It is used for large-scale multiplex analysis for the transmembrane regulatory gene of the disease.

  In a paper by Jou et al. (“Deletion Detection in Dystrophin Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology” Human Mutation 5: 86-93 (1995)), specific regions of multiple exons were amplified and The use of the so-called “gap ligase chain reaction” process, in which amplification products are read on immunochromatographic strips with antibodies specific for different hapten differences on the probe to exons, is described.

  For example, in the field of genetic screening, there is an increasing need for methods useful for detecting the presence or absence of each of a number of sequences contained in a target polynucleotide. For example, cystic fibrosis involves as many as 400 different mutations. Screening for a genetic predisposition to this disease is best done by identifying individuals who are positive for “cystic fibrosis” by examining various variations in the genetic sequence that can be included in the subject's genomic DNA. Ideally it would be a single assay to check for any possible mutation sites. However, it is difficult to apply the above-described prior art method to detection of a specific sequence group by a simple and automated single assay method.

  Solid-phase hybridization assays require multiple liquid processing steps, and some incubation and wash temperatures are carefully controlled to maintain the rigor required to identify single nucleotide mismatches. There must be. Multiplexing of this method is difficult because the optimal hybridization conditions vary greatly depending on the probe sequence.

  Allele-specific PCR products are generally the same size, and any amplification tube is evaluated by the presence or absence of product bands in the gel lane associated with each reaction tube (Gibbs et al., Nucleic Acids Res., 17: 2437-2448 (1989)). Such a method requires the separation of test samples in multiple reaction tubes using various primer combinations, thus increasing the cost of the assay. PCR can also identify alleles in a single reaction tube by attaching various fluorescent dyes to competing allele primers (FFChehab et al., Proc. Natl. Acad. Sci. USA. 86: 9178 ~ 9182 (1989)), but the multiplex analysis by this method is because there are not so many dye species that can be decomposed into spectra by an economical method using existing equipment and knowledge of dye chemistry. Scale expansion is limited. Although the incorporation of bases modified to have large side chains can be used to identify allelic PCR products based on electrophoretic mobility, this method is better suited for modified bases by polymerase. Limited by the incorporation and ability of electrophoresis to degrade relatively large PCR products that differ in the size of one of these groups (Livak et al., Nucleic Acids Res., 20: 4831-4837 (1989)). Each PCR product is used to search for only one mutation and is difficult to multiplex.

  For ligation of allele-specific probes, solid phase capture (U. Landegren et al., Science, 241: 1077-1080 (1988); Nickerson et al., Proc. Natl. Acad. Sci. USA. 87: 8923-8927 (1990) )) Or size-dependent separation methods (DYWu et al., Genomics, 4: 560-569 (1989) and F. Barany, Proc. Natl. Acad. Sci., 88: 189-193 (1991)) It is common to resolve allele signals. However, the latter method is limited in multiplexing because the size of the connecting probe is narrow. The gap ligase chain reaction requires an additional step, ie, extension with a polymerase. When using a probe with a significant friction / resistance ratio accompanying charge / translational motion for more complex multiplexing, it is necessary to lengthen the electrophoresis time and use another detection method.

  Thus, there remains a need for a rapid single assay method for detecting the presence or absence of multiple specific sequences contained in a polynucleotide sample.

Use of Oligonucleotide Arrays for Nucleic Acid Analysis Arrays of ordered oligonucleotides immobilized on a solid support have been proposed for use in DNA sequencing, sorting, isolation and manipulation. It has been observed that hybridization of cloned single-stranded DNA molecules to any oligonucleotide probe of any length can theoretically identify the corresponding complementary DNA segment present in the molecule. . In such arrays, individual oligonucleotide probes are immobilized at various predetermined locations on the solid support. Any oligonucleotide segment in the DNA molecule can be examined using such an array.

  An example of a procedure for sequencing DNA molecules using an oligonucleotide array is disclosed in US Pat. No. 5,202,231 by Drmanac et al. This patent relates to the use of target DNA on a solid support to which a plurality of oligonucleotides are attached. The sequence is read by hybridization of the target DNA segment to the oligonucleotide and the collection of overlapping segments of the oligonucleotide that formed a hybrid. Although any oligonucleotide with a length on the order of 11-22 nucleotides can be used in the array, there is very little information on how to construct this type of array. Regarding this matter, a paper by ABChetverin et al. ("Sequencing of Pools of Nucleic Acids on Oligonucleotide Arrays" BioSystems, 30: 215-31 (1993)), International Publication No. 92/16655 by Khrapko et al. , Kuznetsova et al. (“DNA Sequencing by Hybridization with Oligonucleotides Immbilized in Gel. Chemical Ligation as a Method of Expanding the Prospects for the Method” Mol. Biol. 28 (20); 290-99 (1994)), See also the paper by MALivits et al. ("Dissociation of Duplexes Formed by Hybridization of DNA with Gel-Immobilized Oligonucleotides" J. Biomolec. Struct. & Dynam., 11 (4): 783-812 (1994)). .

  WO 89/10977 by Southern supports with oligonucleotide arrays capable of hybridization reactions used for analysis of nucleic acid samples for known point mutations, genomic fingerprinting, linkage analysis and sequencing The use of the body is disclosed. Nucleotide bases can be placed on a support in a specific pattern to create a matrix. WO 89/10977 describes that a hydroxyl linker can be used for a support having oligonucleotides that are assembled by pen plotter or masking.

  International Publication No. 94/11530 by Cantor also relates to the use of oligonucleotide arrays to perform the sequencing process by hybridization. The oligonucleotide is double-stranded with an overhang, and the target nucleic acid binds to this overhang and is linked to the double-stranded non-overhang. Such arrays are constructed using streptavidin-coated filter paper that captures biotinylated oligonucleotides and are attached after assembly.

  International Publication No. 93/17126 by Chetverin uses a two-component oligonucleotide array for the purpose of classifying and examining nucleic acids. In this array, invariant nucleotide sequences are bound adjacent to the variable nucleotide sequences, and either sequence is bound to the solid support via a covalent bond. The invariant nucleotide sequence has a priming region that allows PCR to amplify the hybridized strand. Next, classification is performed on the variable region by hybridization. Sequencing, isolation, classification, and manipulation of fragmented nucleic acids on a two-component array is also disclosed. In one embodiment with enhanced sensitivity, the immobilized oligonucleotide has a short complementary region that hybridizes therewith leaving a portion of the oligonucleotide uncovered. The array is then subjected to hybridization conditions, and nucleic acids complementary to the immobilized oligonucleotides anneal. DNA ligase is then used to link the short complementary region and the complementary nucleic acid on the array. Little is disclosed about the preparation of oligonucleotide arrays.

  International Publication No. 92/10588 by Fodor et al. Discloses nucleic acid sequencing, fingerprinting and mapping processes by hybridization to oligonucleotide arrays. Oligonucleotide arrays are prepared by the synthesis of polymers immobilized on a very large scale capable of synthesizing large-scale and diverse oligonucleotides. In this method, the substrate surface is put into a functional state, and a linker group for assembling oligonucleotides on the substrate is introduced. In the region where the oligonucleotide is attached, there is a protective group (on the base or on each nucleotide subunit) that is selectively activated. Typically, this method requires imaging the array with light using a variety of mask shapes to deprotect the exposed areas. In the deprotected region, a chemical reaction occurs with the protected nucleotide, and the oligonucleotide sequence is extended to be imaged. By using a two-component masking method, two or more arrays can be constructed in any time. The detection is performed by determining the position of the region where the hybrid is formed. For this, see US Pat. Nos. 5,324,633 and 5,424,186 by Fodor et al., US Pat. Nos. 5,143,854 and 5,405,783 by Pirrung et al., International Publication No. 90/15070 by Pilung, ACPease et al. See also the paper ("Light-generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis" Proc. Natl. Acad. Sci USA 91: 5022-26 (1994)). In a paper by KLBeattie et al. ("Advances in Genosensor Research" Clin. Chem., 41 (5): 700-09 (1995)), a method for binding assembled oligonucleotide probes to a solid support. Explained.

  The above-described sequencing method based on hybridization to an array has many drawbacks. First, it is necessary to synthesize a very large number of oligonucleotides. Second, it is difficult to distinguish correctly hybridized, correspondingly paired duplexes, and mispaired ones. Furthermore, certain oligonucleotides are unlikely to form hybrids under standard conditions, and detailed hybridization conditions must be considered in order for the oligonucleotide to gain identification ability.

  The present invention aims to overcome the disadvantages in the prior art.

SUMMARY OF THE INVENTION The present invention relates to a plurality of capture oligonucleotide probes for use on a support in which a plurality of capture oligonucleotide probes have a narrow range of melting temperatures and complementary oligonucleotides hybridize with little mismatch. On how to design. The first step of the method is (1) each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, and (2) two tetramers complementary to each other are set. A tetramer that is not present in (3) a palindrome or a repeat of 2 nucleotides is not present in the set, and (4) a tetramer having 1 or more or 3 or more nucleotides G or C Providing a first set of tetramers, which are four nucleotides linked together, not present in the set. Groups of 2 to 4 tetramers in the first set are linked together to form a set of multimeric units. From a set of multimeric units, all multimeric units formed from the same tetramer, and all multimeric units having a melting temperature (Celsius) less than 4 times the number of tetramers that form the multimeric unit Are removed to form a modified set of multimeric units. The modified set of multimeric units is arranged in the order of melting temperature in the list. The order of the modified set of multimeric units is randomized within 2 ° C increments of melting temperature. The alternating multimeric units in the list are then divided into first and second subsets, each arranged in order of melting temperature. After reversing the order of the second sub-set, the first set is sequentially connected to the inverted second set to form a set of double multimeric units. In order to form a modified set of double multimeric units, a set of double multimeric units is (1) a melting temperature of less than 11 times the number of tetramers and more than 15 times the number of tetramers ( (2) a double multimer unit in which the same three tetramers are interconnected, and (3) the same four tetramers are interconnected with or without fragmentation Those units with double multimeric units are removed.

Another aspect of the invention involves the support and a collection of double multimeric unit oligonucleotides at different sites on the support so that complementary oligonucleotides immobilized on the solid support are captured at different locations. It relates to an oligonucleotide array. Complementary oligonucleotides are thought to hybridize with little mismatch to members of the assembly of double multimeric unit oligonucleotides within a narrow temperature range above 24 ° C. (1) Each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, (2) no two tetramers complementary to each other are present in the set, and ( 3) A tetramer that is a palindrome or a repeat of 2 nucleotides is not present in the set, a double multimer unit oligonucleotide is formed from the tetramer set, and the assembly of double multimer unit oligonucleotides is then Were removed: (1) oligonucleotides with a melting temperature (Celsius) of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) the same three tetramers Double multimer units interconnected, and (3) multimer units in which the same four tetramers are interconnected with or without fragmentation.

Yet another aspect of the invention relates to a method for identifying one or more sequences that differ by one or more single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences. The method includes providing a sample that potentially includes one or more target nucleotide sequences having a plurality of sequence differences. The plurality of oligonucleotide probe sets also includes (a) a first oligonucleotide probe having a target-specific portion and a positionable array-specific portion, and (b) a target-specific portion and a detectable reporter label. Each set characterized by a second oligonucleotide probe comprising is provided. Oligonucleotide probes in a particular set are suitable to be interconnected when hybridized adjacent to each other on the corresponding target nucleotide sequence, but hybridize to any other nucleotide sequence present in the sample. When soybeans have mismatches that interfere with such ligation. The ligase is also provided with a sample, a plurality of oligonucleotide probe sets, and a ligase that is turbid to form a mixture. A denaturation process in which any hybridizing oligonucleotide is separated from the target nucleotide sequence, and, if present in the sample, each target nucleotide sequence is adjacent to the oligonucleotide probe set in a base-specific manner. To form a ligation product sequence comprising: (a) a positionable array specific portion, (b) a target specific portion linked to each other, and (c) a detectable reporter label The mixture is subjected to one or more ligase detection reaction cycles that include a hybridization process that are linked together. Oligonucleotide probe sets can hybridize to nucleotide sequences present in the sample other than each target nucleotide sequence, but are not ligated together due to the presence of one or more mismatches and are individually To be separated. The capture oligonucleotide has a nucleotide sequence that is complementary to the positionable array-specific portion and is supported by different capture oligonucleotides immobilized at different sites, formed from a collection of double multimeric unit oligonucleotides Provided to the body. Oligonucleotides with an array-specific portion that can be localized hybridize with little mismatch to members of the collection of capture oligonucleotides within a narrow temperature range of more than four times the number of tetramers in the multimer unit. I think that. (1) Each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, (2) no two tetramers complementary to each other are present in the set, and ( 3) A tetramer that is a palindrome or a repeat of 2 nucleotides forms a double multimeric unit oligonucleotide from a tetramer set that is not present in the set. In order to form a modified set of double multimeric units, the set of double multimeric unit oligonucleotides has then removed the following oligonucleotides: (1) less than 11 times the number of tetramers and four An oligonucleotide with a melting temperature (Celsius) greater than 15 times the number of monomers, (2) a double multimer unit in which the same three tetramers are linked together, and (3) the same four tetramers Double multimer units interconnected with or without fragmentation. After subjecting the mixture to one or more ligase detection reaction cycles, the positionable array-specific portion hybridizes to the capture oligonucleotide probe in a base-specific manner, thereby allowing the positionable array-specific portion. However, the mixture is contacted with the support under conditions effective to be captured on the support at sites having complementary capture oligonucleotides. The reporter label of the ligation product captured on the support at a specific site is detected, indicating that one or more target nucleotide sequences are present in the sample.

Another aspect of the invention relates to a kit for identifying one or more sequences that differ by single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences. In addition to ligase, the kit is suitable for the oligonucleotide probes in a particular set to be linked together when hybridized adjacent to each other on each target nucleotide sequence, but others present (A) a first oligonucleotide probe having a target sequence-specific portion and a positionable array-specific portion, and (b) having a mismatch that prevents such ligation when hybridizing to the nucleotide sequence of A plurality of oligonucleotide probe sets, each characterized by a second oligonucleotide probe comprising a target sequence specific portion and a detectable reporter portion. The capture oligonucleotide has a nucleotide sequence complementary to a positionable array-specific portion and has different capture oligonucleotides immobilized at different sites formed from a collection of double multimeric unit oligonucleotides A support is also found in the kit. Oligonucleotides with an array-specific portion that can be localized hybridize with little mismatch to members of the collection of capture oligonucleotides within a narrow temperature range of more than four times the number of tetramers in the multimer unit. Conceivable. (1) Each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, (2) no two tetramers complementary to each other are present in the set, and ( 3) From the tetramer set, double multimeric unit oligonucleotides are formed, where tetramers that are palindromic or 2 nucleotide repeats are not present in the set. The collection of double multimer unit oligonucleotides then removes the following oligonucleotides: (1) Number of tetramers, where the capture oligonucleotide has a nucleotide sequence that is complementary to a positionable array specific portion An oligonucleotide having a melting temperature (Celsius) less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) a double multimeric unit in which the same three tetramers are linked together, and (3) A double multimeric unit in which the same four tetramers are interconnected with or without fragmentation.

  Another aspect of the invention relates to a method that avoids synthesizing ligase detection reaction oligonucleotides that would be improperly cross-hybridized to capture oligonucleotides on a solid support. The method includes comparing the ligase detection reaction oligonucleotide to a capture oligonucleotide and identifying any capture oligonucleotide that is likely to cross-hybridize with the ligase detection reaction oligonucleotide.

Detailed Description of the Invention and Drawings The present invention provides a plurality of capture oligonucleotide probes for use on a support having a narrow range of melting temperatures and to which complementary oligonucleotides hybridize with little mismatch. It relates to a method of designing a capture oligonucleotide probe. The first step of the method is (1) each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, and (2) two tetramers complementary to each other are set. A tetramer that is not present in (3) a palindrome or a repeat of 2 nucleotides is not present in the set, and (4) a tetramer having 1 or more or 3 or more nucleotides G or C Providing a first set of tetramers, which are four nucleotides linked together, not present in the set. Groups of 2 to 4 tetramers in the first set are linked together to form a set of multimeric units. From a set of multimeric units, all multimeric units formed from the same tetramer, and all multimeric units having a melting temperature (Celsius) less than 4 times the number of tetramers that form the multimeric unit Are removed to form a modified set of multimeric units. The modified set of multimeric units is arranged in the order of melting temperature in the list. The order of the modified set of multimeric units is randomized within 2 ° C increments of melting temperature. The alternating multimeric units in the list are then divided into first and second subsets, each arranged in order of melting temperature. After reversing the order of the second sub-set, the first set is sequentially connected to the inverted second set to form a set of double multimeric units. In order to form a modified set of double multimeric units, a set of double multimeric units is (1) a melting temperature of less than 11 times the number of tetramers and more than 15 times the number of tetramers ( (2) a double multimer unit in which the same three tetramers are interconnected, and (3) the same four tetramers are interconnected with or without fragmentation Those units with double multimeric units are removed.

Another aspect of the invention involves the support and a collection of double multimeric unit oligonucleotides at different sites on the support so that complementary oligonucleotides immobilized on the solid support are captured at different locations. It relates to an oligonucleotide array. Complementary oligonucleotides are thought to hybridize with little mismatch to members of the assembly of double multimeric unit oligonucleotides within a narrow temperature range above 24 ° C. (1) Each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, (2) no two tetramers complementary to each other are present in the set, and ( 3) A tetramer that is a palindrome or a repeat of 2 nucleotides is not present in the set, a double multimer unit oligonucleotide is formed from the tetramer set, and the assembly of double multimer unit oligonucleotides is then Were removed: (1) oligonucleotides with a melting temperature (Celsius) of less than 12.5 times the number of tetramers and more than 14 times the number of tetramers, (2) the same three tetramers Double multimer units interconnected, and (3) multimer units in which the same four tetramers are interconnected with or without fragmentation.

Yet another aspect of the invention relates to a method for identifying one or more sequences that differ by one or more single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences. The method includes providing a sample that potentially includes one or more target nucleotide sequences having a plurality of sequence differences. The plurality of oligonucleotide probe sets also includes (a) a first oligonucleotide probe having a target-specific portion and a positionable array-specific portion, and (b) a target-specific portion and a detectable reporter label. Each set characterized by a second oligonucleotide probe comprising is provided. Oligonucleotide probes in a particular set are suitable to be interconnected when hybridized adjacent to each other on the corresponding target nucleotide sequence, but hybridize to any other nucleotide sequence present in the sample. When soybeans have mismatches that interfere with such ligation. The ligase is also provided with a sample, a plurality of oligonucleotide probe sets, and a ligase that is turbid to form a mixture. A denaturation process in which any hybridizing oligonucleotide is separated from the target nucleotide sequence, and, if present in the sample, each target nucleotide sequence is adjacent to the oligonucleotide probe set in a base-specific manner. To form a ligation product sequence comprising: (a) a positionable array specific portion, (b) a target specific portion linked to each other, and (c) a detectable reporter label The mixture is subjected to one or more ligase detection reaction cycles that include a hybridization process that are linked together. Oligonucleotide probe sets can hybridize to nucleotide sequences present in the sample other than each target nucleotide sequence, but are not ligated together due to the presence of one or more mismatches and are individually To be separated. The capture oligonucleotide has a nucleotide sequence that is complementary to a positionable array-specific portion and is formed from a collection of double multimer unit oligonucleotides and is supported by a capture oligonucleotide immobilized on different sites. Provided to. Oligonucleotides with an array-specific portion that can be localized hybridize with little mismatch to members of the collection of capture oligonucleotides within a narrow temperature range of more than four times the number of tetramers in the multimer unit. I think that. (1) Each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, (2) no two tetramers complementary to each other are present in the set, and ( 3) A tetramer that is a palindrome or a repeat of 2 nucleotides forms a double multimeric unit oligonucleotide from a tetramer set that is not present in the set. In order to form a modified set of double multimeric units, the set of double multimeric unit oligonucleotides has then removed the following oligonucleotides: (1) less than 11 times the number of tetramers and four An oligonucleotide with a melting temperature (Celsius) greater than 15 times the number of monomers, (2) a double multimer unit in which the same three tetramers are linked together, and (3) the same four tetramers Double multimer units interconnected with or without fragmentation. After subjecting the mixture to one or more ligase detection reaction cycles, the positionable array-specific portion hybridizes to the capture oligonucleotide probe in a base-specific manner, thereby allowing the positionable array-specific portion. However, the mixture is contacted with the support under conditions effective to be captured on the support at sites having complementary capture oligonucleotides. The reporter label of the ligation product captured on the support at a specific site is detected, indicating that one or more target nucleotide sequences are present in the sample.

Another aspect of the invention relates to a kit for identifying one or more sequences that differ by single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences. In addition to ligase, the kit is suitable for the oligonucleotide probes in a particular set to be linked together when hybridized adjacent to each other on each target nucleotide sequence, but others present (A) a first oligonucleotide probe having a target sequence-specific portion and a positionable array-specific portion, and (b) having a mismatch that prevents such ligation when hybridizing to the nucleotide sequence of A plurality of oligonucleotide probe sets, each characterized by a second oligonucleotide probe comprising a target sequence specific portion and a detectable reporter portion. The capture oligonucleotide has a nucleotide sequence complementary to a positionable array-specific portion and has different capture oligonucleotides immobilized at different sites formed from a collection of double multimeric unit oligonucleotides A support is also found in the kit. Oligonucleotides with an array-specific portion that can be localized hybridize with little mismatch to members of the collection of capture oligonucleotides within a narrow temperature range of more than four times the number of tetramers in the multimer unit. Conceivable. (1) Each tetramer in the set differs from all other tetramers in the set by at least 2 nucleotide bases, (2) no two tetramers complementary to each other are present in the set, and ( 3) From the tetramer set, double multimeric unit oligonucleotides are formed, where tetramers that are palindromic or 2 nucleotide repeats are not present in the set. The collection of double multimer unit oligonucleotides then removes the following oligonucleotides: (1) Number of tetramers, where the capture oligonucleotide has a nucleotide sequence that is complementary to a positionable array specific portion An oligonucleotide having a melting temperature (Celsius) less than 11 times the number of tetramers and more than 15 times the number of tetramers, (2) a double multimeric unit in which the same three tetramers are linked together, and (3) A double multimeric unit in which the same four tetramers are interconnected with or without fragmentation.

  Often, multiple different single base mutations, insertions or deletions may occur at the same nucleotide position of the target sequence. This method comprises a set of oligonucleotides wherein the second oligonucleotide probe comprises a common detectable label and the first oligonucleotide probe has a different positionable array specific part and a target specific part Including the use of The first oligonucleotide probe is preferred for ligation to the second adjacent oligonucleotide probe at the first linking junction when it hybridizes to the test sequence without mismatch. Different first contiguous oligonucleotide probes contain different discriminating bases at the junction site, and only hybridization without mismatches can be linked at the junction position. Each first contiguous oligonucleotide contains a portion specific for a different positionable array, so that specific base changes are identified by capture at different addresses. In this scheme, a plurality of different capture oligonucleotides are attached to different positions on a solid support for multiplex detection of another nucleic acid sequence that differs from other nucleic acids by at least one base. Alternatively, the first oligonucleotide probe includes a portion that is specific to a common positionable array, and the second oligonucleotide probe has a different detectable label and a target-specific portion.

  Such an arrangement enables multiplex detection of another nucleic acid sequence having at least one base different from other nucleic acids. In carrying out such multiplex detection, the nucleic acid sequences are present on the same or different alleles.

  The invention also relates to kits for carrying out the methods of the invention including ligases, sets of different oligonucleotide probes, and solid supports on which capture oligonucleotides are immobilized. Primers used for preliminary amplification of the target nucleotide sequence are also included in the kit. If amplification is performed by polymerase chain reaction, a polymerase is also included in the kit.

  1 and 2 show a flow chart of the method of the present invention compared to a prior art ligase detection reaction using capillary electrophoresis or gel electrophoresis / fluorescence quantification. FIG. 1 relates to germline mutation detection and FIG. 2 illustrates cancer detection.

  FIG. 1 shows the detection of point mutations in the germline, such as the p53 mutation, that causes Li-Fraumenisyndrome. In step 1, after preparation of the DNA sample, exons 5-8 were PCR amplified using Taq (ie, Thermusaquaticus) polymerase under hot start conditions. At the end of the reaction, Taq polymerase is inactivated by treatment with proteinase K. In step 2, the product is diluted 20-fold with fresh LDR buffer containing allele-specific and common LDR probes. Tubes typically contain about 500 femtomole of each primer. In stage 3, the ligase detection reaction is initiated under hot start conditions by the addition of Taq ligase. LDR probes are ligated to adjacent probes only in the presence of target sequences that exhibit complete complementarity at the junction site. The product is detected in two different forms. In the first form 4a, the fluorescently labeled LDR probe used in the prior art comprises different lengths of poly A or hexaethylene oxide tails. Thus, each LDR product resulting from ligation to normal DNA with slightly different mobility gives a peak ladder. Germline mutations generate new peaks in the electrophoretic image. The size of the new peak roughly corresponds to the amount of mutation present in the original sample. 0% for normal homozygotes, 50% for heterozygous carriers, and 100% for homozygous mutants. In the second form 4b, according to the present invention, each allele-specific probe comprises, for example, an additional 24 nucleotide bases at its 5 ′ end. These sequences are single positionable sequences that hybridize specifically to complementary address sequences on the positionable array. In an LDR reaction, each allele-specific probe can be linked to an adjacent fluorescently labeled common probe in the presence of the corresponding target sequence. Wild type and mutant alleles are captured at adjacent addresses on the array. Wash off unreacted probe. Black dots indicate 100% signal for the wild type allele. White dots indicate 0% signal for mutant alleles. Dotted dots indicate 50% signal for each allele at one position in germline mutations.

  FIG. 2 represents detection of somatic mutations in the p53 tumor suppressor gene, both of which are usually related to low sensitivity mutation detection. In step 1, a DNA sample is prepared and exons 5-9 are amplified as 3 fragments by PCR using fluorescent PCR primers. This makes it possible to quantify the fluorescence of the PCR product using capillary electrophoresis or gel electrophoresis in step 2. In stage 3, the product spikes at 1/100 dilution of marker DNA (for each of the three fragments). This DNA is homologous to wild-type DNA except that it contains mutations not observed in cancer cells, but is easily detected with a suitable LDR probe. The mixed DNA product from step 4 is diluted 20-fold with a buffer containing all LDR probes specific only to the mutant or marker allele. In step 5, the ligase detection reaction was initiated under hot start conditions by the addition of Taq ligase. Only in the presence of a target sequence that gives complete complementarity at the junction site, ligation of the LDR probe to the adjacent probe occurs. The product is detected in the same two forms described in FIG. In the form used in the prior art of step 6a, the products are separated by capillary or gel electrophoresis and the fluorescent signal is quantified. The ratio of mutant peak to marker peak gives an estimate of the amount of cancer mutation present in the original sample divided by 100. In the form of step 6b, according to the present invention, the product is detected by specific hybridization to complementary sequences on a positionable array. The fluorescence signal ratio of the mutant dot to the marker dot gives an estimate of the amount of cancer mutation present in the original sample divided by 100.

The method of the detection reaction by the ligase of the present invention is best understood by referring to FIGS. In general, this is described in Barany et al., International Publication No. 90/17239; F. Barany et al., “Cloning, Overexpression and Nucleotide Sequence of Cloning, Overexpression and Nucleotide Sequence of Genes Encoding Thermostable DNA Ligase. a Thermostable DNA Ligase-encoding Gene) ( Gene. 109: 1-11 (1991)) and F. Barany et al., “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase” Amplification Using Cloned Thermostable Ligase ”( Proc. Natl. Acad. Sci. USA. 88: 189-193 (1991)) and the like, the disclosures of which are incorporated herein by reference. Two sets of complementary oligonucleotides can be used in the detection reaction by the ligase of the present invention. Fins are known as the ligase chain reaction described in the three references listed immediately above and are incorporated herein by reference. Alternatively, the detection reaction with ligase involves a single cycle known as an oligonucleotide ligation assay. Landegren et al. “A Ligase-Mediated Gene Detection Technique” Science 241: 1077-80 (1988); Landegren et al. “DNA Diagnostics-Molecular Techniques and Automation” Science 242: 229-37 (1988); and Landegren et al., US Pat. No. 4,988,617.

  During the detection reaction phase of the method with ligase, hybridization occurs at 50-85 ° C, while denaturation is performed at 80-105 ° C. Each cycle consists of a denaturation treatment and a heat hybridization treatment as a whole for about 1-5 minutes. Usually, the detection reaction by ligation involves 2 to 50 cycles of repeated denaturation and hybridization. The total time required for the detection reaction step by ligase of the method is 1 to 250 minutes.

  The set of oligonucleotide probes includes ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleotide analogs, modified peptide nucleotide analogs, modified phosphate / sugar backbone oligonucleotides, nucleotide analogs, and mixtures thereof It may be a form.

  In one variation, the oligonucleotides of the set of oligonucleotide probes each have a hybridization or melting temperature (ie, Tm) of 66-70 ° C. These oligonucleotides are 20-28 nucleotides in length.

It is desirable to chemically or enzymatically destroy unconverted LDR oligonucleotide probes containing nucleotide array-specific portions that can be located before capturing the ligation product on the DNA array. If this is not done, such unconverted probes will compete with the ligation product for binding at addresses on the array of solid supports containing complementary sequences. The disruption utilizes exonucleases such as exonuclease III and others (LH Guo and R. Wu, Methods in Enzymology 100: 60-96 (1985), which is incorporated herein by reference), blocked at the ends and This can be achieved by combining with an LDR probe that is not included in probe ligation. The blocking moiety can contain a reporter group or a phosphorothioate group (TTNikiforow et al., “Use of Phosphorothioate Primer and Exonuclease Hydrolysis for the Preparation of Single-Strand PCR Products and Detection by Solid Phase Hybridization (The Use of Phosphorothioate Primers and Exonuclease Hydrolysis for the Preparation of Single-stranded PCR Products and their Detection by Solid-phase Hybridization), PCR Methods and Applications. 3: p.285-291 (1994), which is incorporated herein by reference. ). After the LDR method, probes that have not been ligated are selectively destroyed by reacting the reaction mixture with exonuclease. The ligated probe is protected by removal of the free 3 'end necessary to initiate the exonuclease reaction. This approach results in an increase in signal to noise ratio, especially when the LDR reaction forms only a small amount of product. Since oligonucleotides that have not been ligated compete with the capture oligonucleotide for capture, competition with such ligated oligonucleotides reduces the signal. Another advantage of this approach is that sequences containing unhybridized labels are degraded, thus making it difficult to generate a target-independent background signal. This is because it can be more easily removed from the DNA array by washing.

  The above set of oligonucleotide probes has a preferred reporter label for detection. Useful labels include chromophores, fluorescent moieties, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent sites, and electrochemical detection sites. Capture oligonucleotides are in the form of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, peptide nucleotide analogs, modified peptide nucleotide analogs, modified phosphate-sugar backbone oligonucleotides, nucleotide analogs, and mixtures thereof. possible. If the method of the invention involves the use of multiple oligonucleotide sets, the second oligonucleotide probe may be the same, but the site specific for the positionable array of the first oligonucleotide probe is different. Alternatively, the site specific to the positionable array of the first oligonucleotide probe may be the same, but the reporter label of the second oligonucleotide probe is different.

Prior to the detection reaction phase by the ligation of the present invention, the sample is preferably amplified by the first target nucleic acid amplification method. This increases the amount of the target base sequence in the sample. For example, amplification of the initial target nucleic acid is accomplished using polymerase chain reaction, sequence self-replication, or RNA amplification mediated by Q-β replicase. The polymerase chain reaction method is a preferred amplification method and is described in H. Erlich et al., “Recent Advances in the Polymerase Chain Reaction” ( Science 252: 1643-50 (1991); M. Innis et al. "PCR protocol: A Guide to Methods and Applications" (Academic Press: New York (1990)); and R. Saiki et al., "Primer-specific enzymatic of DNA with thermostable DNA polymerase." Amplification (Primer-directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase) "( Science 239: 487-91 (1988)) and the like, which are incorporated herein by reference. J. Guatelli et al., “Isothermal, invitro Amplification of Nucleic Acids by a Multienzyme Reaction Mod, modeled on retroviral replication. eled After Retroviral Replication ”( Proc. Natl. Acad. Sci. USA 87: 1874-78 (1990)) describes a method for self-replication of sequences. RNA amplification mediated by Qβ replicase is described in F. Kramer et al., “Replicatable RNA Reporters” ( Nature 339: 401-02 (1989)), which is incorporated herein by reference.

  The use of the polymerase chain reaction method of the present invention and the subsequent detection method with ligase is shown in FIG. Here the homozygosity or heterozygosity (ie allelic difference) in the two polymorphisms is on the same gene. Alternatively, such allelic differences may be on different genes.

  As shown in FIG. 3, when the target nucleic acid is in the form of a double-stranded DNA molecule, it is denatured to separate the strands. This is accomplished by heating to a temperature on the order of 80 to 105 ° C. Next, a polymerase chain reaction primer is added and hybridized to the strand, usually at a temperature of about 20 to 85 ° C. In addition, a thermostable polymerase (for example, Thermosaquaticus polymerase) is added, the temperature is adjusted to 50 to 85 ° C., and the primer is extended along the length of the nucleic acid to which the primer hybridizes. After the extension step of the polymerase chain reaction, the resulting double-stranded molecule is heated to a temperature of 80 to 105 ° C. to denature the molecule and separate the strands. These hybridization, extension, and denaturation steps are repeated many times to amplify the target to the appropriate level.

  When the polymerase chain reaction phase of the method is completed, the detection reaction phase by ligation shown in FIG. 3 is started. In the case of a double-stranded DNA molecule, the target nucleic acid is denatured at a temperature of 80 to 105 ° C., preferably 94 ° C., and then an oligonucleotide probe for detection reaction by ligation to one strand of the target base sequence is ligated. (For example, heat-resistant ligase-like Thermusaquaticus ligase shown in FIG. 3). Next, the oligonucleotide probe is hybridized to the target nucleic acid molecule, and ligation is usually performed at a temperature of 45-85 ° C., preferably 65 ° C. If the ligation junction is perfectly complementary, the oligonucleotides can be ligated together. When the substitutable nucleotide is T or A, if T is present in the target base sequence, the oligonucleotide probe having the site Z1 specific to the localizable array is linked to the oligonucleotide probe having the reporter label F. If A is present in the target base sequence, the oligonucleotide probe having the site Z2 specific to the position-identifiable array is linked to the oligonucleotide probe having the reporter label F. Similarly, when the variable nucleotide is A or G, the oligonucleotide probe having the site Z4 specific to the positionable array is the oligonucleotide probe having the reporter label F if T is present in the target base sequence. In addition, if C is present in the target base sequence, an oligonucleotide probe having a site Z3 specific to a position-identifiable array is linked to an oligonucleotide probe having a reporter label F. After ligation, the material is denatured again to separate the hybridized strands. The hybridization / ligation and denaturation steps amplify the target signal through one or more cycles (eg, 1-50 cycles). Fluorescent ligation products (also non-ligated oligonucleotide probes with sites specific to the localizable array) are the Z1, Z2, Z3, and Z4 sites at specific addresses on the localizable array Captured by hybridization to a complementary capture probe. Next, the presence of the ligated oligonucleotide is detected with a pre-existing label F on one of the oligonucleotides. In FIG. 3, the sequence of the ligation product is a positionable array that hybridizes to the address of the array with capture oligonucleotides complementary to sites Z1 and Z3 specific for the positionable array, but no ligation has occurred. An oligonucleotide probe having specific sites Z2 and Z4 hybridizes to its complementary capture oligonucleotide. However, only the sequences of the ligation products have the label F, so their presence is detected.

  In FIG. 4, FIG. 4 is similar to FIG. 3, except that the common oligonucleotide probe has a position-specific portion while the allele-specific probe has a different label.

  FIG. 5 is a flow chart relating to the PCR / LDR stage of the present invention, and it is possible to identify any given base at any site. The emergence of fluorescent signals at addresses complementary to sites Z1, Z2, Z3, and Z4 specific to the localizable array indicates the presence of A, G, C, and T alleles in the target sequence, respectively. Show. Here, the presence of the target base sequences A and C alleles is indicated by address fluorescence on the solid support with capture oligonucleotide probes complementary to sites Z1 and Z3, respectively. Note that in FIG. 5, the site specific to the localizable array is on the discriminating oligonucleotide probes and the discriminating base is at the 3 ′ end of these probes.

  In FIG. 6, FIG. 6 is similar to FIG. 5 except that the common oligonucleotide probe has a position-specific portion while the allele-specific probe has a different label.

  FIG. 7 is a flow chart of the PCR / LDR stage for detecting the presence of possible bases at two adjacent sites of the present invention. Here, the LDR primers may overlap, but can be linked when the junction has complete complementarity. This is the difference in LDR from other approaches such as allele-specific PCR where overlapping primers inhibit each other. In FIG. 7, the first nucleotide position is heterozygous for the A and C alleles, while the second nucleotide position is heterozygous for the G, C and T alleles. As shown in FIG. 5, the site specific to the localizable array is on the discriminating oligonucleotide probe and the discriminating base is at the 3 ′ end of these probes. A reporter group (eg, a fluorescent label) is at the 3 ′ end of the common oligonucleotide probe. For example, if each individual has two normal genes and two pseudogenes, and there are three possible single bases (G, A, and C) at two splice sites (nucleotide 656) This is possible for the 21 hydroxylase gene. It can also be used to detect low level mutations in HIV infection that indicate the emergence of drug resistant (eg, against AZT) strains. Returning to FIG. 7, the appearance of fluorescent signals with addresses complementary to sites Z1, Z2, Z3, Z4, Z5, Z6, Z7, and Z8 specific to the localizable array The presence of A, G, C, and T, respectively, in the A and C alleles, and the presence of A, G, C, and T, respectively, in the G, C, and T alleles at the heterozygote site are indicated.

  In FIG. 8, FIG. 8 is similar to FIG. 7, except that the common oligonucleotide probe has a position-specific portion while the allele-specific probe has a different label.

  FIG. 9 is a flow chart for the PCR / LDR stage of the present invention, identifying insertions (upper left probe set) and deletions (lower right probe set). On the left, the normal sequence contains 5 As in the poly A tail. The mutant sequence has two additional A's inserted in the tail. Thus, LDR products with sites Z1 (representing normal sequence) and Z3 (representing a 2 base pair insertion) specific to the localizable array are fluorescently labeled by ligation to a common primer. LDR methods (eg, using thermostable ligase enzymes) can easily identify single base insertions or deletions in single nucleotide repeats, but allele-specific PCR cannot distinguish such differences. This is because the 3 ′ base is present in both alleles. On the right, the normal sequence is 5 (CA) repeats (ie CACACACACA). Variants contain 2 fewer CA bases compared to the normal sequence (ie CACACA). These are detected as fluorescent LDR products at specific sites Z8 (representing the normal sequence) and Z6 (representing two CA deletions) addresses specific to the localizable array. Also, the resistance of various infectious agents to drugs can be examined using the present invention. In FIG. 9, the presence of the sequence of the ligation product indicated by the fluorescent label F at the address with capture oligonucleotides complementary to Z1 and Z3 indicates the presence of both normal and mutant poly A sequences. Similarly, the presence of the sequence of the ligation product indicated by the fluorescent label F of the address with capture oligonucleotides complementary to Z6 and Z8 is both the normal CA repeat sequence and the sequence lacking one repeat unit. Indicates existence.

  In FIG. 10, FIG. 10 is similar to FIG. 9 except that the common oligonucleotide probe has a position-specific portion while the allele-specific probe has a different label.

  FIG. 11 is a flow chart for the PCR / LDR stage of the present invention using a site specific to a positionable array that detects low level mutations in the presence of excess normal sequence. FIG. 11 shows the sequence GGT of codon 12 encoding glycine (Gly) of the K-ras gene. Only a few cells contain a G to A mutation in GAT that encodes aspartic acid (Asp). Wild type LDR probes (ie, normal sequences) are excluded from the reaction. When normal LDR probes (with discriminating base = G) are included, they can be strengthened over the signal derived from the mutant target by linking them to a common probe. Alternatively, as shown in FIG. 11, the presence of the sequence of the ligation product with fluorescent label F indicates that aspartic acid at the address having a capture oligonucleotide complementary to the site Z4 specific for the positionable array. Indicates the presence of the encoding variant.

  In FIG. 12, FIG. 12 is similar to FIG. 11 except that the common oligonucleotide probe has a position specific portion while the allele specific probe has a different label.

  FIG. 13 is a flow chart for the PCR / LDR stage of the present invention, where a site specific to a positionable array is located on a common oligonucleotide probe, and the identification oligonucleotide probe has a reporter label. Allelic differences are distinguished by different fluorescent signals F1, F2, F3, and F4. This method allows the use of higher density arrays. This is because each position is predicted to emit light with a specific group. It has the disadvantage of requiring fluorescent groups that have minimal overlap in the emission spectrum and require multiple scanning. This is not ideally suited for detecting low levels of alleles (eg, mutations associated with cancer).

  FIG. 14 is a flow chart for the PCR / LDR stage of the present invention, in which both flanking and nearby alleles are detected. Adjacent mutations are right next to each other, and a set of oligonucleotide probes identifies the base at the 3 'end of the junction (sites Z1, Z2, Z3, and Z4 specific for different positionable arrays However, the other set of oligonucleotide probes identifies the base at the 5 ′ end of the junction (use of different fluorescent reporter labels F1, F2, F3, and F4). In FIG. 14, the codons encoding Gly and arginine (Arg) of the disease gene (eg, cystic fibrosis CFTR) are each a candidate for germline mutation. The detection results in FIG. 14 show that Gly (GGA; indicated by site Z2 with the sequence of the ligation product and labeled F2) is glutamic acid (Glu) (GAA; indicated by sequence of the ligation product with site Z2 and label F1). Mutated and Arg (CGG; indicated by sequence of ligation product with site Z7 and labeled F2) in tryptophan (Trp) (TGG; indicated by sequence of ligation product with site Z8 and label F2) Indicates that a mutation has occurred. Thus, the patient is a compound heterozygous individual (ie, having mutant alleles in both genes).

  FIG. 15 is a flow chart for the PCR / LDR stage of the present invention, in which all possible single base variations for a single codon are detected. Since the amino acid codon is usually degenerate at the third base, the first two positions can be determined at the protein level for all these possible mutations. These amino acids include arginine, leucine, serine, threonine, proline, alanine, glycine, and valine. However, for some amino acids, all three bases of the codon are examined, so an oligonucleotide probe that distinguishes mutations at three adjacent positions is required. As shown in Figure 15, we designed 4 oligonucleotide probes containing 4 possible bases in the penultimate position relative to the 3 'end and another containing 4 possible bases at the 3' end. Four capture oligonucleotides are designed to solve this problem. A common oligonucleotide probe having only a reporter label corresponds to codon degeneracy and has two fluorescent groups that can be captured at the same array address and discriminated between sequences of different ligation products. For example, as shown in FIG. 15, the presence of codons encoding glutamine (Gln) (ie, CAA and CAG) is indicated by the presence of the sequence of the ligation product containing site Z1 and label F2. Similarly, the presence of Gln for a mutation encoding histidine (His) (encoded by codon CAC) indicates the presence of the sequence of the ligation product with site Z1 and label F2, and the sequence of the ligation product with site Z7 and label F2. Indicated by the presence of. Due to the fact that primers Z1 and Z7 have the same sequence, redundancy is inherent in this assay.

  A particularly important aspect of the present invention is that the amount of target base sequence in a sample can be quantified. By obtaining an internal (ie, standard material obtained by amplification and detection using the sample) or an external (ie, the resulting standard is not amplified but detected using the sample), this is Can be achieved in a way.

  According to a certain quantitative method, the signal generated by the reporter label is generated and detected by capturing the sequence of the ligation product produced from the analytical sample. This signal intensity is compared with a calibration curve obtained by the signal generated by the capture of the sequence of ligation products present in a sample having a known amount of target base sequence generated. As a result, the amount of the target base sequence in the analysis sample can be examined. The technology involves the use of external standards.

  Another quantification method of the invention relates to an internal standard. Here, a known amount of one or more marker target base sequences is added to the sample. In addition, a set of marker specific oligonucleotide probes is added to the mixture along with the ligase, the set of oligonucleotide probes and the sample. The set of marker-specific oligonucleotide probes has (1) a target-specific site complementary to the marker target base sequence as well as a site specific to the localizable array complementary to the capture oligonucleotide on the support. A first oligonucleotide probe, (2) a second oligonucleotide probe having a target-specific site complementary to the marker target base sequence and a detectable reporter label. Oligonucleotide probes in a particular marker specific oligonucleotide set are suitable in hybridizing adjacent to each other on the corresponding marker target base sequence and ligating together. However, when hybridizing to other nucleotide sequences or added marker sequences in the sample, there are mismatches that inhibit such ligation. The presence of the sequence of the ligation product captured on the support is identified by detection of the reporter label. Next, the amount of target base sequence in the sample is determined by comparing the amount of ligation product generated and captured from a known amount of marker target base sequence with the amount of sequence of other captured ligation products. .

  Another quantification method of the present invention includes analysis of a sample containing two or more target base sequences having a plurality of sequence differences. Here, the sequence of the ligation product corresponding to the target base sequence is detected and identified by any of the above techniques. Next, the relative amount of the sequence of the ligation product generated and captured is compared, and the relative amount of the target base sequence in the sample is quantified. This provides a quantitative measure of the relative level of the target base sequence in the sample.

Prior to the detection reaction method step using the ligase of the present invention, the ligase chain reaction method may be performed to amplify the oligonucleotide product. This method is described by F. Barany et al., “Cloning, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-encoding Gene”, Gene 109: 1-11 (1991), and F. Barany et al., “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase” ( Proc. Natl. Acad. Sci. USA 88 : 189-93 (1991)), which are incorporated herein by reference. Instead of using ligase chain reaction for amplification, transcription-based amplification methods can also be used.

A preferred thermostable ligase is from Thermusaquaticus. The enzyme can be isolated from this organism (M. Takahashi et al., “Thermophillic DNA ligase” ( J. Biol. Chem. 259: 10041-47 (1984)); this is hereby incorporated by reference. Incorporated). Alternatively, it can be prepared with a recombinant. Such isolation methods include the production of recombinant Thermusaquaticus ligase (and Thermosthemophilus ligase), as well as Barany et al., International Publication No. 90/17239, and F. Barany et al., “Cloning of a gene encoding a thermostable DNA ligase, Overexpression and Nucleotide Sequence of a Thermostable DNA Ligase-encoding Gene "( Gene. 109: 1-11 (1991)) and the like, which are incorporated herein by reference. These documents contain complete sequence information of this ligase and the DNA encoding it. Other preferred ligases include E. coli ligase, T4 ligase, Thermussp.AK16 ligase, Aquifexaeolicus ligase, Thermotogamaritima ligase and Pyrococcus ligase.

  The ligated amplification mixture may contain carrier DNA such as salmon sperm DNA.

  In the hybridization stage, which is preferably a heat hybridization treatment, discrimination between base sequences based on identification nucleotides at the ligation junction is performed. Differences between target base sequences are, for example, single nucleobase differences, nucleic acid deletions, and rearrangements. Such sequence differences including one or more bases can also be detected. Preferably, the set of oligonucleotide probes have substantially the same length and hybridize to the target base sequence under substantially similar hybridization conditions. As a result, the method of the present invention can detect infectious diseases, genetic diseases, and cancer. It is also useful in environmental monitoring, forensic science, and food science.

  A wide variety of infectious diseases can be detected by the method of the present invention. These are usually caused by bacterial, viral, parasitic, and fungal infectious agents. Also, the resistance of various infectious agents to drugs can be examined using the present invention.

  Bacterial infectious agents detected in the present invention include Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacteria Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasturella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, yellow Staphylococcus aureus, Staphylococcus pneumonia, B-Hemolyticstrep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Ureaplasma Chlamydia (Chlam ydia, Neisseriagonorrhea, Neisserimeningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis (Proteus helicobacter) Includes pylori, Treponema palladium, Borreliaburgdorferi, Borrelia recurrentis, Rickettsial pathology, Nocardia, and Acitnomycetes.

  Fungal infectious agents detected in the present invention include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, para Coccionioides brasiliensis, Candidaalbicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Spiis thrix ), And Maduromycosis.

  Viral infectious agents detected in the present invention include human immunodeficiency virus, human T cell lymphocyte affinity virus, hepatitis virus (eg, hepatitis B virus and hepatitis C virus), Epstein Barr virus, cytomegalo Including viruses, human papillomaviruses, orthomyxoviruses, paramyxoviruses, adenoviruses, coronaviruses, rhabdoviruses, polioviruses, togaviruses, vanyaviruses, arenaviruses, rubella viruses, and reoviruses.

  Parasitic acting pathogens detected in the present invention include Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchovervavolvulus, Leishmania, Trypanosoma species, Schistosoma species, Entamoebahistolytica, Cryptosporidum, Giardia species, Giardia species (Trichimonas), Balatidiumcoli, Wuchereriabancrofti, Toxoplasma sp., Enterobiusvermicularis, Ascarislumbricoides, Trichu Trichuristrichiura, Dracunculusmedinesis, trematodes, Diphyllobothriumlatum, Taenia species, Pneumocystiscarinii, and Necatoramericanis )including.

  The present invention is also useful for detecting drug resistance by infectious agents. For example, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, Mycobacterium tuberculosis, and AZT-resistant human immunodeficiency virus are all included in the present invention. Can be identified.

  Genetic diseases can be detected by the method of the present invention. This can be done by prenatal screening for chromosomal and genetic abnormalities or postnatal screening for genetic diseases. Examples of detectable genetic diseases include 21 hydroxylase deficiency, cystic fibrosis, fragile X syndrome, Turner syndrome, Duchenne muscular dystrophy, Down's syndrome or other trisomy, heart disease, single gene disease, HLA typing, phenyl ketone Examples include urine disease, sickle cell anemia, Tay-Sachs disease, thalassemia, Kleinfelder syndrome, Huntington's disease, autoimmune disease, fat metabolism disorder, obesity, hemophilia, inborn errors of metabolism, and diabetes.

  The cancer detected by the method of the present invention usually includes an oncogene, a tumor suppressor gene, or a gene involved in DNA amplification, replication, recombination, or repair. Examples of these include BRCA1 gene, p53 gene, familial colon polyposis, Her2 / Neu amplification, Bcr / AbI, K-ras gene, human papillomavirus types 16 and 18, leukemia, colon cancer, breast cancer, lung cancer, prostate cancer, Includes brain tumors, central nervous system tumors, bladder tumors, melanoma, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian cancers, otorhinolaryngological tumors, and loss of heterozygosity.

  In the field of environmental monitoring, the present invention relates to pathogenicity and presence in natural and engineered ecosystems such as municipal wastewater purification systems and reservoirs and in contaminated areas that are purifying microecosystems or biological environments. Used for the detection, identification and monitoring of microorganisms. Detection of plasmids containing genes that metabolize non-biological substances, either for monitoring specific target microorganisms in population dynamics studies, or for genetic detection, identification, or monitoring in modified microorganisms in environmental and industrial plants It is also possible to do it.

  The invention can also be used in a variety of forensic fields, including military and criminal human identification, paternity testing and family relationship analysis, HLA compatibility typing, and blood, sperm, or transplant organ contamination screening. .

  In the food and feed industry, the present invention has a wide variety of applications. For example, it can be used for identification and characterization of production microorganisms such as beer, wine, cheese, yogurt, bread and other yeasts involved in production. Another area of use relates to contamination in product quality control and verification and construction methods (eg livestock, sterilization, and meat processing). Other uses include characterization of plants, bulbs and seeds for breeding purposes, identification of the presence of plant-specific pathogens, and detection and identification of livestock infections.

  Desirably, oligonucleotide probes are preferred for ligation at ligation junctions when hybridizing adjacent to each other on corresponding target sequences based on perfect complementarity at the ligation junction. However, when the oligonucleotide probes in the set hybridize to any other nucleotide sequence in the sample, there is a mismatch in the base at the ligation junction that inhibits ligation. Most preferably, the mismatch is present at the base adjacent to the 3 ′ base of the ligation junction. Alternatively, the mismatch may be in a base adjacent to the base at the junction junction.

  According to the method of the present invention, the first and second base sequences can be detected in a sample in an amount of 100 to 250 femtomole. By coupling the LDR step with a first polymerase specific amplification step, the complete method of the invention can detect the target base sequence in a sample containing as much as a single molecule. Furthermore, PCR amplification products that are often in picomolar amounts can be easily diluted within the above range. In this ligase detection reaction, the rate of formation of the mismatched ligation product sequence is less than 0.005 of the rate of formation of the matched ligation product sequence.

  Once the concatenation period is complete, the capture period begins. In the capture phase of the method, the mixture is contacted with the solid support at a temperature of 25-90 ° C., preferably 60 ° C.-80 ° C., and for 10-180 minutes, preferably up to 60 minutes. Hybridization can be accelerated by mixing the ligation mixture during hybridization or by adding volume exclusion, chaotropic agents, tetramethylammonium salts, or N, N, N, trimethylglycine (betaine monohydrate) or Improved. If the array consists of tens to hundreds of addresses, it is important to have the opportunity for the correct ligation product to hybridize at the appropriate address. This can be accomplished by thermal motion of the oligonucleotide at the high temperature used, mechanical motion of the liquid in contact with the array surface, or motion of the oligonucleotide across the array by an electric field. To remove unhybridized probes after hybridization and optimize the detection of captured probes, the array is washed with buffer. Alternatively, the array is washed sequentially.

  Hybridization specificity may be facilitated by the addition of non-specific competitor DNA (eg, herring sperm DNA) and / or the addition of formamide to the hybridization solution. Wash stringency can also be increased by increasing the wash temperature and / or adding formamide to the wash buffer. FIG. 16 shows the results from various combinations of the above changes to standard hybridization and wash conditions.

  Preferably, the solid support has a porous surface of a hydrophilic polymer composed of a combination of acrylamide and a carboxylate, aldehyde, or functional monomer containing an amino group. This surface is formed by coating the support with a gel based on polyacrylamide. Preferred formulations are acrylamide / acrylic acid and N, N-dimethylacrylamide / glycerol monomethacrylate. The crosslinker N, N′-methylenebisacrylamide is at a level of 50: 1 or less, preferably 500: 1 or less.

One aspect related to masking negative charges while contacting a solid support with a ligation mixture is achieved through the use of divalent cations. It may be a divalent cation, Mg ²⁺ , Ca ²⁺ , MN ²⁺ , or Co ²⁺ . Usually, masking with divalent cations is performed by pre-hybridizing the solid support with a hybridization buffer containing cations at a minimum concentration of 10 mM for 15 minutes at room temperature.

  Another embodiment for masking negative charges while contacting a solid support with a ligation mixture is performed by contacting at a pH of pH 6.0 or lower. It is advantageous to add a buffer to the ligation mixture before or during contact with the solid support. A preferred buffer comprises 2- (N-morpholino) ethanesulfonic acid (MES), sodium phosphate, and potassium phosphate.

  Another embodiment for masking negative charges while the solid support is in contact with the ligation mixture is performed by capping free carboxylic acid groups with a neutralizing agent while reducing the hydrophilicity of the polymer. Preferred neutralizing agents include ethanolamine, diethanolamine, propanolamine, dipropanolamine, isopropanolamine, and diisopropanolamine. Masking with a neutralizing agent is usually performed by activating carboxylic acid groups in a solid support with 1-[(3-dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, followed by chloroform. , By treatment with a neutralizer solution in a polar aprotic solvent such as dichloromethane or tetrahydrofuran.

  Negative charge masking according to the present invention improves detection of the presence of ligated products in the presence of oligonucleotide probes that have not been ligated. In particular, the detection of the presence of a ligation product when the ratio to oligonucleotide probes that have not been ligated is less than 1: 300, preferably less than 1: 900, more preferably less than 1: 3000, most preferably less than 1: 9000. In addition, the present invention is effective.

  Furthermore, the masking of negative charges according to the present invention improves the ability to discriminate and detect the presence of a target base sequence having a target base sequence different from the non-target nucleotide sequence by a single base difference. In particular, for detection of a target base sequence when the ratio of target base sequence to non-target nucleotide sequence is less than 1:20, preferably less than 1:50, more preferably less than 1: 100, and most preferably less than 1: 200. The present invention is effective.

  The selection of capture oligonucleotides and positionable base sequences that hybridize stably is important. For this purpose, the oligonucleotide set and capture oligonucleotide are designed to hybridize to the target base sequence at a temperature below the temperature at which the capture oligonucleotide hybridizes to a site specific to the positionable array. There is a need. If oligonucleotides are not designed in this way, false positive signals are generated by capturing adjacent unreacted oligonucleotides from the same set of oligonucleotides that hybridize to the target.

  If ligation of a particular oligonucleotide set occurs and the ligation correlates with the presence or absence of the target base sequence in the test sample, the detection phase of the method includes scanning and identification. Scanning can be performed by scanning electron microscopy, confocal microscopy, charge coupled devices, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, conductivity, and fluorescence or phosphorescent imaging techniques. Correlation is performed by a computer.

  Another aspect of the present invention relates to a method for forming an array of oligonucleotides on a support. The method includes providing a support having an array of sites, each of which is suitable for oligonucleotide addition. A preferred linker or support (preferably non-hydrolyzable) for attachment of the oligonucleotide to the support at each array position is attached to the solid support. An array of oligonucleotides on a solid support is formed by a series of cycles of activation of selected array positions for the addition of multimeric nucleotides and addition of multimeric nucleotides at the active array positions.

  Yet another aspect of the invention relates to the oligonucleotide array itself on the support. The support has an array of sites that are each suitable for the addition of oligonucleotides. A preferred linker or support (preferably non-hydrolyzable) for binding the oligonucleotide to the support is attached to the support at each position of the array. The array of oligonucleotides is disposed at least in some array positions occupied by oligonucleotides having 16 nucleotides or more on the support.

  In array formation methods, multimeric oligonucleotides from different multimeric oligonucleotide sets bind at different array locations on the support. As a result, the support includes an array of positions having different groups of multimeric oligonucleotides attached to different positions.

  The 1,000 different addresses may be unique capture oligonucleotides (eg, 24 mer) covalently linked to the target specific sequence (eg, about 20-25mer) of the LDR oligonucleotide probe. The sequence of the capture oligonucleotide probe is one that does not show any homology to either the target sequence on the genome or other sequences present in the sample. The oligonucleotide probe is then captured in a site specific to the localizable array, a sequence complementary to the capture oligonucleotide on the localizable support array. This concept is shown in two possible forms, for example, detection of the p53R248 mutation (FIGS. 17A-17C).

  FIGS. 17A-17C show two other forms for designing oligonucleotide probes to identify the presence of a germline mutation at codon 248 of the p53 tumor suppressor gene. The wild type sequence encodes arginine (R248), but in cancer mutations it encodes tryptophan (R248W). The lower figure is a schematic diagram of the capture oligonucleotide. A thick horizontal line represents a membrane or surface containing a positionable array. A thin curve indicates a flexible linker arm. The thicker line shows the capture oligonucleotide sequence bound to the solid surface in the C to N direction. For illustrative purposes, the capture oligonucleotide is drawn vertically such that the linker arm in Part B is “stretched”. Since the arms are flexible, base-pair complementarity allows capture oligonucleotides to hybridize 5 'to C and 3' to N in each. When the oligonucleotide binds to the membrane at the N-terminus, oligonucleotide hybridization in the same direction is possible. In this case, DNA / PNA hybridization is usually 5 'to 3' and 3 'to 5' antiparallel. Other modified sugar phosphate backbones are used as well. FIG. 17B shows two LDR probes designed to distinguish wild-type and mutant p53 by including a base C or T discrimination at the 3 ′ end. In the presence of the correct target DNA and Tth ligase, the discriminating probe binds covalently to the downstream common oligonucleotide. The downstream oligonucleotide is fluorescently labeled. Identification oligonucleotides are distinguished by the presence of sites Z1 and Z2 specific for a single positionable array at each 5 ′ end. Black dots indicate that target-dependent ligation is occurring. After ligation, the oligonucleotide probe may be captured by a site specific to a complementary positionable array at a single address on the array. Both ligated and unreacted oligonucleotide probes are captured by the oligonucleotide array. Unreacted fluorescently labeled common primer and target DNA are washed away at high temperature (about 65 ° C. to 80 ° C.) and low salt concentration. Mutant signals are identified by detection of fluorescent signals in capture oligonucleotides complementary to the site-specific array Z1, while wild-type signals are complementary to the site-specific array Z2. Appear in typical capture oligonucleotides. Heterozygosity is indicated by comparable signals in the capture oligonucleotides complementary to sites Z1 and Z2 specific for the localizable array. The signal is quantified using a fluorescence imaging device. This form uses a single address for each allele and is suitable for achieving very accurate detection of low levels of signal (30-100 attomole LDR product). FIG. 17C shows that the identification signal can be quantified using a fluorescence imaging apparatus. In this form, a single address is used that identifies the oligonucleotide probe by having different fluorescent groups at the 5 ′ end, F1 and F2. Any of the oligonucleotide probes can be linked to a common downstream oligonucleotide probe that contains a site Z1 specific for the 3′-endable array. In this form, both wild type and mutant LDR are captured at the same address on the array and are distinguished by different fluorescence. This form allows for more favorable use of the array and is suitable for detecting hundreds of potential germline mutations.

  The support can be made from a wide variety of materials. The substrate may be biological, non-biological, organic, inorganic, or any combination thereof, particles, chains, precipitates, gels, sheets, tubular materials, spheres, containers, capillaries, pads, slices, films, It exists as a plate, slide, disk, membrane, etc. The substrate has a normal shape such as a board, a square, a circle, or the like. The substrate is preferably flat, but may take a variety of different surface configurations. For example, the substrate may have raised or depressed areas where synthesis occurs. The substrate and its surface preferably form a rigid support on which to carry out the reactions described herein. Also, the substrate and its surface are selected to provide suitable light absorption properties. For example, the substrate can be polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, or a wide variety of gels, or (poly) tetrafluoroethylene, (poly) vinylidene Polymers such as difluoride, polystyrene, polycarbonate, polyethylene, polypropylene, polyvinyl chloride, poly (methyl acrylate), poly (methyl methacrylate), or combinations thereof may be used. Those skilled in the art can easily understand other matrix materials from the description of the present disclosure. In a preferred embodiment, the substrate is flat glass or single crystal silicon.

  In some embodiments, the substrate surface is etched using known techniques to provide the desired surface properties. For example, trenches, V-grooves, hill structures, rounded platforms, etc. are used, and the synthesis region is a reflection “mirror” structure that maximizes the concentration of light from fluorescent sources. Place it closer to the focal point.

  The surface on the substrate is usually but not always composed of the same material as the substrate. Thus, the surface is composed of any of a wide variety of materials such as polymers, plastics, ceramics, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or combinations thereof. The surface is functionalized with a binding member firmly bonded to the substrate surface. Preferably, the functional group on the surface is a reactive group such as silanol, olefin, amino, hydroxyl, aldehyde, keto, halo, acyl halide or carboxyl group. In some cases, such functional groups are pre-existing in the substrate. For example, silica-based materials have silanol groups, polysaccharides have hydroxyl groups, and synthetic polymers contain a variety of functional groups depending on the monomer that produces them. Alternatively, if the substrate does not contain the desired functional group, such groups can be attached to the substrate in one or more steps.

A variety of commercially available materials can be used, including suitably modified glass, plastic, or carbohydrate surfaces or various membranes. Depending on the material, surface functional groups (eg silanol, hydroxyl, carboxyl, amino) are present from the outset (possibly as part of the coating polymer) or another method for the introduction of functional groups (For example, plasma animation, chromic acid oxidation, functionalized side chain treatment with alkyltrichlorosilane is required). The hydroxyl group is incorporated into a stable carbamic acid (urethane) linkage in several ways. The amino functional group can be directly acetylated, while the carboxyl group is activated (eg, N, N′-carbodiimidazole or water soluble carbodiimide) and reacted with an amino functionalized compound. As shown in FIG. 18, the support may be a membrane or surface having a starting functional group X. The transformation of the functional group can be done in a variety of ways (where appropriate) providing a group X ^* that represents one partner in a covalent bond with the group Y ^* . FIG. 18 illustrates the grafting of PEG (ie, polyethylene glycol), but the same repertoire of reactions can be carried out with carbohydrates (with hydroxyl), linkers (with carboxyl), and / or preferred functional groups (amino or carboxyl). ) Can be used to add the extended oligonucleotide (if necessary). In some cases, the group X ^* or Y ^* is preactivated (a species that can be isolated in another reaction). Alternatively, activation occurs in situ. Referring to the PEG described in FIG. 18, Y and Y ^* may be the same (identical binary functional group) or different (heterogeneous binary functional group). In the latter case, Y can be protected for further chemical control. Unreacted amino groups are blocked by acetylation and succinylation to maintain a neutral or negatively charged environment that “eliminates” excess unhybridized DNA. The load level can be examined by standard analytical methods. Fields et al. "Principles and Practice of Solid-Phase Peptide Synthesis" (Svnthetic Peptides: A User's Guide) G. Grant, Editor, WH Freeman and Co .: New York. P. 77-183 (1992)), which is incorporated herein by reference.

  One approach to adding functional groups to a silica-based support surface includes molecules with the desired functional group (eg, olefin, amino, hydroxyl, aldehyde, keto, halo, acyl halide or carboxyl), or the desired Examples include silanization with any molecule A that can bind to another molecule B containing a functional group. In the former case, the functionalization of the support based on glass or silica, for example with amino groups, is performed by 3-aminopropyltriethoxysilane, 3-aminopropylmethyldiethoxysilane, 3-aminopropyldimethylethoxysilane, 3-aminopropyltriethoxysilane, Aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropylmethyldimethoxysilane, N- (2-aminoethyl-3-aminopropyl) trimethoxysilane, aminophenyltrimethoxysilane, 4-aminobutyl It is carried out with an amine compound such as dimethylmethoxysilane, 4-aminobutyltriethoxysilane, aminoethylaminomethylphenethyltrimethoxysilane, or a mixture thereof. In the latter case, molecule A preferably contains an olefin group such as vinyl, acrylate, methacrylate or allyl, while molecule B contains an olefin group and the desired functional group. In this case, molecules A and B polymerize with each other. In some cases, to modify its properties (eg, to impart biocompatibility and improve mechanical stability). It is preferred to modify the silanized surface. This can be achieved by adding an olefin molecule C along with molecule B to obtain a polymer network comprising molecules A, B, and C.

式中、R¹はHもしくはCH₃である。
R²は（C=O）-O-R⁶で、官能置換基を有するもしくは有さない脂肪族基、官能置換基を有するもしくは有さない芳香族基、または官能置換基を有するもしくは有さない脂肪族/芳香族基の混合したものである。
R³はO-アルキル、アルキルもしくはハロゲン基である。
R⁴は、O-アルキル、アルキルもしくはハロゲン基である。
R⁵はO-アルキル、アルキルもしくはハロゲン基であり、また
R⁶は、官能置換基を有するもしくは有さない脂肪族基、官能置換基を有するもしくは有さない芳香族基、または官能置換基を有するもしくは有さない脂肪族/芳香族基の混合したものである。分子Aの例は、3-（トリメトキシシリル）プロピルメタクリレート、N-[3-（トリメトキシシリル）プロピル］-N'-（4-ビニルベンジル）エチレンジアミン、トリエトキシビニルシシラン、トリエチルビニルシシラン、ビニルトリクロロシシラン、ビニルトリメトキシシラン、およびビニルトリメチルシシランを含む。Molecule A is defined by the following chemical formula:

In the formula, R ¹ is H or CH ₃ .
R ² is (C═O) —OR ⁶ , an aliphatic group with or without a functional substituent, an aromatic group with or without a functional substituent, or a fat with or without a functional substituent A mixture of aromatic / aromatic groups.
R ³ is an O-alkyl, alkyl or halogen group.
R ⁴ is an O-alkyl, alkyl or halogen group.
R ⁵ is an O-alkyl, alkyl or halogen group, and
R ⁶ is an aliphatic group with or without a functional substituent, an aromatic group with or without a functional substituent, or a mixture of aliphatic / aromatic groups with or without a functional substituent. It is. Examples of molecule A are 3- (trimethoxysilyl) propyl methacrylate, N- [3- (trimethoxysilyl) propyl] -N '-(4-vinylbenzyl) ethylenediamine, triethoxyvinylsilane, triethylvinylsilane , Vinyltrichlorosilane, vinyltrimethoxysilane, and vinyltrimethylsilane.

式中、（i）R¹はHもしくはCH₃であり、
R²は（C=O）、および
R³はOHもしくはClである；
または
（ii）R¹はHもしくはCH₃、およびR²は（C=O）-O-R⁴、官能置換基を有するもしくは有さない脂肪族基、官能置換基を有するもしくは有さない芳香族基、または官能置換基を有するもしくは有さない脂肪族/芳香族基の混合したものである。また、R³はOH、COOH、NH₂、ハロゲン、SH、COCl、もしくは活性エステル等の官能基、およびR4は官能置換基を有するもしくは有さない脂肪族基、官能置換基を有するもしくは有さない芳香族基、または官能置換基を有するもしくは有さない脂肪族/芳香族基の混合したものである。分子Bの例は、アクリル酸、アクリルアミド、メタクリル酸、ビニル酢酸、4-ビニル安息香酸、イタコン酸、アリルアミン、アリルエチルアミン、4-アミノスチレン、2-アミノエチルメタクリレート、塩化アクリロイル、塩化メタクリロイル、クロロスチレン、ジクロロスチレン、4-ヒドロキシスチレン、ヒドロキシメチルスチレン、ビニルベンジルアルコール、アリルアルコール、2-ヒドロキシエチルメタクリラート、もしくはポリ（エチレングリコール）メタクリレートを含む。Molecule B is a monomer containing one or more functional groups as described above. Molecule B is defined by the following chemical formula:

In which (i) R ¹ is H or CH ₃
R ² is (C = O), and
R ³ is OH or Cl;
Or (ii) R ¹ is H or CH ₃ , and R ² is (C═O) —OR ⁴ , an aliphatic group with or without a functional substituent, an aromatic group with or without a functional substituent Or a mixture of aliphatic / aromatic groups with or without functional substituents. R ³ has a functional group such as OH, COOH, NH ₂ , halogen, SH, COCl, or an active ester, and R 4 has an aliphatic group with or without a functional substituent, or has a functional substituent. Or a mixture of aliphatic / aromatic groups with or without functional substituents. Examples of molecule B are acrylic acid, acrylamide, methacrylic acid, vinyl acetic acid, 4-vinyl benzoic acid, itaconic acid, allylamine, allylethylamine, 4-aminostyrene, 2-aminoethyl methacrylate, acryloyl chloride, methacryloyl chloride, chlorostyrene , Dichlorostyrene, 4-hydroxystyrene, hydroxymethylstyrene, vinyl benzyl alcohol, allyl alcohol, 2-hydroxyethyl methacrylate, or poly (ethylene glycol) methacrylate.

  Molecule C is a molecule polymerizable to molecule A, molecule B, or both, and optionally includes one or more functional groups as described above. Molecule C includes acrylic acid, methacrylic acid, vinyl acetic acid, 4-vinylbenzoic acid, itaconic acid, allylamine, allylethylamine, 4-aminostyrene, 2-aminoethyl methacrylate, acryloyl chloride, methacryloyl chloride, chlorostyrene, dichlorostyrene, 4-hydroxystyrene, hydroxymethylstyrene, vinylbenzyl alcohol, allyl alcohol, 2-hydroxyethyl methacrylate, poly (ethylene glycol) methacrylate, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, styrene, 1-vinylimidazole, 2-vinylpyridine, 4-vinylpyridine, divinylbenzene, ethylene glycol dimethacrylate acrylate, N, N'-methylenediacrylamide, N, N'-phenylenediacrylamide, 3 , 5-Bis (acryloylamide) benzoic acid, pentaerythritol triacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetraacrylate, trimethylolpropane ethoxylate (14 / 3EO / OH) triacrylate, trimethylolpropane ethoxylate (7 / 3EO / OH) triacrylate, triethylolpropane propoxylate (1PO / OH) triacrylate, or a monomer such as trimethylolpropane propoxylate (2PO / PH triacrylate) or a crosslinker may be used.

  Usually, the functional group serves as a starting point for the oligonucleotide that ultimately binds to the support. These functional groups can be reactive to organic groups attached to the support or can be modified to be reactive to such groups through the use of linkers or handles. Functional groups can impart various desired properties to the support.

  After functionalization of the support (optional), a tailor-made polymer network containing activated functional groups that serve as carrier sites for complementary oligonucleotide capture probes is implanted into the support. Thus, the advantage of this approach is that the loading capacity of the capture probe can be significantly increased and the physical properties of the solid phase versus the liquid phase as an intermediate can be easily controlled. Parameters subject to optimization include the type and concentration of monomers containing functional groups, and the type and relative concentration of crosslinkers used.

  Although it is understood that a linker molecule is not a necessary element of the present invention, a functionalized substrate surface is preferably provided with a linker molecule layer. The linker molecule is preferably of sufficient length so that in the prepared substrate the polymer can freely interact with the molecule exposed to the substrate. The linker molecule needs to be 6-50 atoms long so that it is fully exposed. Linker molecules such as aryl acetylene, ethylene glycol oligomers containing 2 to 10 monomer units, diamines, divalent acids, amino acids, or combinations thereof.

  In another embodiment, linker molecules are selected based on their hydrophilic / hydrophobic nature to improve the presentation of synthetic polymers to specific receptors. For example, in the case of a hydrophilic receptor, the hydrophilic linker molecule is preferably such that the receptor can be closer to the synthetic polymer.

  In another alternative embodiment, the linker molecule is provided as having a photocleavable group at an intermediate position. The photocleavable group is preferably cleaved at a wavelength different from that of the protecting group. This allows for the removal of various polymers after synthesis is completed by exposure to different light wavelengths.

  The linker molecule is bonded to the substrate with a bond between carbons, for example using a (poly) trifluorochloroethylene surface, or preferably with a siloxane bond (for example using a glass or silicon oxide surface). it can. In one embodiment, the siloxane bond with the substrate surface is formed by the reaction of a linker molecule having a trichlorosilyl group. The linker molecule may be selectively attached to an aligned array (ie, as if its heads are flat in a polymerized monolayer). In another embodiment, the linker molecule is adsorbed on the substrate surface.

As an example of assembling an array using multimers, such assembly can be accomplished using tetramers. Of the 256 (4 ⁴ ) possible methods in which ⁴ bases can be arranged as a tetramer, 36 with a unique sequence can be selected. Each selected tetramer differs from all others by at least 2 bases, and no two dimers are complementary to each other. Furthermore, tetramers thought to lead to address self-pairing or hairpin formation were removed.

  The final tetramers are listed in Table 1 and are arbitrarily numbered from 1 to 36. This unique tetramer set is sometimes used as a design module for the desired 24mer capture oligonucleotide address sequence. It is believed that the structure can be assembled by stepwise (one base at a time) or convergent (tetramer building block) synthesis strategy. Many other tetramer sets that follow the above rules may be designed. It is believed that the segmentation method is not limited to tetramers only, but other units can be used, ie dimers, trimers, pentamers or hexamers.

[Table 1] List of tetrameric PNA sequences and complementary DNA sequences that differ from each other by at least 2 bases

Note that in the tetramer numbering scheme, each address is abbreviated with six numbers (eg, Table 2, second column below). From the concept of 24mer addresses (Table 1) designed with a single set of 36 tetramers, the number of possible structures is vast (36 ⁶ = 2,176,782,336).

  FIG. 19 shows one of many possible designs with 36 tetramers differing from each other by at least 2 bases. The checkerboard pattern shows 256 possible total tetramers. The square represents the first two bases on the left on the checkerboard followed by the upper two bases. Each tetramer must be at least two bases different from each other and non-complementary. The tetramer is indicated by a white box and its complementary strand is listed as (number). Thus, in this scheme, the complementary sequences GACC (20) and GGTC (20 ′) are mutually exclusive. Furthermore, the tetramer must be non-palindromic, eg TCGA (black diagonal box), and non-repeating, eg CACA (black diagonal box, from top left) Bottom right). All other sequences that differ from the 36 tetramer by only one base are shaded in light gray. Four potential tetramers (white boxes) were not selected because they are either either A * T or all G * C bases. However, as shown below, it is possible to raise the Tm value of A * T base to approximately the level of G * C base. Thus, tetramers (including those in white boxes) that are all A * T or all G * C bases can potentially be used for tetramer design. In addition, thymine can be replaced with 5-propynyluridine provided it is a site specific to the sequence of the capture oligonucleotide address and to the positionable array of oligonucleotide probes. This increases the Tm of A * T base pairs by about 1.7 ° C. Thus, the Tm value for individual tetramers should be about 15.1 ° C to 15.7 ° C. The full length 24mer Tm value should be above 95 ° C.

  For the purpose of illustrating this concept, a subset of 6 of 36 tetramer sequences was used and the arrays: 1 = TGCG; 2 = ATCG; 3 = CAGC; 4 = GGTA; 5 = GACC; and 6 = Constructed ACCT. This single tetramer set can be used as an address sequence design module for the site specific to the desired 24mer positionable array and the 24mer complementary capture oligonucleotide. This embodiment involves the synthesis of five site-specific arrays specific sites (sequences listed in Table 2). Note that in the tetramer numbering scheme, each site abbreviation (referred to as “Zip #”) is indicated by six numbers (referred to as “Zip code”).

[Table 2] List of all 5 DNA / PNA oligonucleotide address sequences

  Each of these oligomers has a hexaethylene oxide linker arm at the 5 ′ end (P. Grossman et al., Nucleic Acids Res., 22: 4527-4534 (1994) is incorporated herein by reference), and a glass slide surface Contains the final amino functional group, which is suitable for attachment to, or another substance. The attachment method depends on free surface functional groups (Y. Zhang et al., Nucleic Acids Res., 19: 3929-3933 (1991) and Z. Guo et al., Nucleic Acids Res., 34: 5456-5465 (1994). Are incorporated herein by reference).

  Synthetic oligonucleotides (either normal and complementary orientation, either capture hybridization or hybridization / ligation) are prepared as either DNA or PNA with either natural bases or nucleotide analogs. Such analogs are perfectly complementary and pair with natural bases, but the Tm value is increased (eg, 5-propynyluracil).

  In accordance with the present invention, false hybridization signals resulting from DNA synthesis errors are avoided. Addresses can be designed such that there is a very large difference in hybridization Tm values for incorrect addresses. In contrast, direct hybridization methods rely on small differences. The present invention also eliminates the problem of erroneous data interpretation using gel electrophoresis or capillary electrophoresis due to either DNA synthesis errors, band expansion, or pseudo-band migration.

  The use of capture oligonucleotides to detect the presence of ligation products eliminates the need to detect single base differences in oligonucleotides using differential hybridization. Other existing methods of the prior art that rely on allele-specific PCR, differential hybridization or sequencing by hybridization must optimize the hybridization conditions individually for each new sequence to be analyzed. Don't be. When attempting to detect multiple mutations simultaneously, it becomes difficult or impossible to optimize hybridization conditions. In contrast, the present invention is a general method that accurately detects the correct signal under uniform hybridization conditions, independent of the target sequence. The present invention creates a flexible method of discriminating between different oligonucleotide sequences with significantly higher accuracy than by any method currently available within the prior art.

  The arrays of the present invention are universal and useful for detecting cancer mutations, hereditary (germline) mutations, and infectious diseases. Further benefits come from the ability to reuse the array, reducing the cost per sample.

  The present invention also provides great flexibility in the synthesis of the oligonucleotide and its anchoring to the support. Oligonucleotides can be synthesized away from the support and then attached to a unique surface on the support. Oligonucleotide multimeric segments that do not require backbone protection of intermediate inclusions (eg, PNA) can be synthesized and linked onto a solid support. Further advantages are achieved by being able to integrate these synthetic methods with the design of capture oligonucleotide addresses. Such fabrication of solid supports is amenable to automated fabrication and obviates the need for human intervention and the resulting contamination concerns.

  An important advantage of the arrays of the present invention is that the arrays can be reused with previously attached capture oligonucleotides. In order to prepare a solid support for such reuse, the captured oligonucleotide must be removed without removing the linking component that links the captured oligonucleotide to the solid support. . Various means are available to achieve this goal. For example, the solid support is treated in distilled water at 95 ° C to 100 ° C, subjected to 0.01 N sodium hydroxide at room temperature, contacted with 50% dimethylformamide at 90 ° C to 95 ° C, or 50 at 90 ° C to 95 ° C. It can be treated with% formamide. In general, this means can be used to remove captured oligonucleotides in about 5 minutes. These conditions are suitable for disrupting DNA-DNA hybridization. DNA-PNA hybridization requires other disruption conditions.

  The present invention is illustrated by the following examples without being limited thereto.

EXAMPLES Example 1 Materials and Methods Oligonucleotide Synthesis and Purification Oligonucleotides were obtained as custom synthesis products from IDT (Coralville, Iowa) or using standard phosphoramidite chemistry (PE) Synthesized in-house on Biosystems, Foster City, Calif.). Spacer phosphoramidite 18, 3'-amino-modifying group C3 CPG and 3'-fluorescein CPG were purchased from Glen Research (Sterling, VA). All other reagents were purchased from PE Biosystems. Both labeled and unlabeled oligonucleotides were purified by 12% denaturing polyacrylamide gel electrophoresis. Bands were visualized by UV shadowing, excised from the gel and eluted overnight at 37 ° C. in 0.5 M NaCl, 5 mM EDTA, pH 8.0. The oligonucleotide solution is purified on C18 Sep-Paks (Waters Corporation, Milford, Mass.) According to the manufacturer's instructions and subsequently concentrated until the oligonucleotide is dry (Speed-Vac) ) And -20 ° C.

Microscope slide cleaning Concentrated NH ₄ OH-30% H ₂ O ₂ -H ₂ O (1: 1: 5, v / v) glass microscope slide (VWR, precleaned, 3 in. × 1 in. × 1.2 mm) Incubated in v / v) for 10 minutes and washed with distilled water. The second incubation was performed in boiling concentrated HCl-30% H ₂ O ₂ —H ₂ O for 10 minutes. U. Jonsson et al., “Absorption Behavior of Fibronectin on Well Characterized Silica Surfaces”, J. Colloid Interface Sci. 90: 148—incorporated herein by reference. 163 (1982). Slides were thoroughly washed with distilled water, methanol and acetone and air dried at room temperature.

Polymer-coated slides Immediately following cleaning, slides (Fisher Scientific, pre-cleaned, 3 in. X 1 in. X 1.2 mm), 2% methacryloxypropyltrimethoxysilane, 0.2% in CHCl ₃ Soaked in triethylamine for 30 minutes at 25 ° C. and then washed with CHCl ₃ (twice 15 minutes). Monomer solution (20μL: 8% acrylamide, 2% acrylic acid, 0.02% N, N'-methylene-bisacrylamide (monomer: crosslinker ratio is 500: 1 ratio), 0.8% ammonium persulfate radical polymerization started Agent) was attached to one end of the slide and spread using a cover slip (24 × 50 mm) that had been previously silanized (5% (CH ₃ ) ₂ SiCl _{2 in} CHCl ₃ ). Polymerization was accomplished by heating the slide on a 70 ° C. hot plate for 4.5 minutes. The slide was removed from the hot plate and the cover slip was immediately peeled off using a single cutting razor blade. Coated slides were washed with deionized water, dried in open air and stored under ambient conditions.

Preparation of Zip-Code Arrays Polymer-coated slides are 1- [3- (dimethylamino) propyl] which is 0.1M in 0.1MK ₂ HPO ₄ / KH ₂ PO ₄ , pH 6.0 It was activated in advance by immersing it in a solution of -3-ethylcarbodiimide hydrochloride with 20 mM N-hydroxysuccinimide added at 25 ° C. for 30 minutes. The activated slide was washed with water and then dried in a 65 ° C. oven. They were stable on storage for 6 months or longer at 25 ° C. in a desiccator with Drierite.

Array is Cartesian Technologies Pixsys 5500 robot using 0.2 μM Zip ₂ oligonucleotide solution in 0.2 MK ₂ HPO ₄ / KH ₂ PO ₄ , pH 8.3 at 25 ° C. and 70% relative humidity. It was spotted at. Each address was spotted in quadruplicate. In addition, Cy3, Cy5 and fluorescein standards were printed along the right hand top and bottom of each array. Following spotting, the unbound oligonucleotide is polymerized by immersing the slide in 300 mM bicine, pH 8.0, 300 mM NaCl, 0.1% SDS at 65 ° C. for 30 minutes, washing with water, and drying. Removed from the surface. Arrays were stored at 25 ° C in slide boxes until needed.

PCR amplification of K-ras DNA samples
PCR amplification consisted of 10 mM Tris HCl, pH 8.3, 4 mM MgCl ₂ , 50 mM KCl, 800 μM dNTPs, 1 μM forward and reverse primers (each primer 50 pmol, K-rasEx1 forward and K-rasEx1 reverse (Table 3)), 1 U It was performed under paraffin oil in a 50 μL reaction mixture containing 100 ng of genomic DNA extracted from Ampli Taq Gold and parafine embedded tumors or cell lines. The reaction was preincubated at 95 ° C. for 10 minutes. Amplification was achieved by 40 rounds of 94 ° C. for 30 seconds, 60 ° C. for 1 minute, and 72 ° C. for 1 minute temperature cycle followed by 72 ° C. for 5 minutes final extension. After PCR, 1 μL of proteinase K (18 mg / mL) was added, and the reaction was heated at 70 ° C. for 10 minutes, and then the reaction was stopped at 95 ° C. for 15 minutes. To confirm the presence of the expected size amplification product, 2 μL of each PCR product was analyzed on a 3% agarose gel.

LDR of K-ras DNA sample
LDR consists of 20 mM Tris HCl, pH 8.5, 5 mM MgCl ₂ , 100 mM KCl, 10 mM DTT, 1 mM NAD ⁺ , 10 pmol total LDR probe (500 fmol of each fluorescence labeled identification probe (Cy3, Cy5 and fluorescently labeled K-rasc32Wt, Cy3 Labeled K-rasc12.2D, Cy5 labeled K-rasc12.2A, fluorescent labeled K-rasc12.2V, Cy3 labeled K-rasc12.1S, Cy5 labeled K-rasc12.1R, fluorescent labeled K-rasc12.1C, Cy3 labeled K -rasc13.4D) with a total of 5 pmol of common probe (each 1500 fmol of K-rascd32Com9cZip1, K-rascd12Com2cZip2, and K-rascd12Com1cZip3, and 500 fmol of K-rascd13Com4cZip4 (Table 3)), 2 μL from cell lines or tumor samples The reaction mixture was run under paraffin oil in a 20 μL volume containing the PCR product, the reaction mixture was pre-heated at 94 ° C. for 2 minutes, and then 25 fmol of wild-type TthDNA ligase was added. 20 round cycles at 65 ° C for 4 minutes.

Hybridization of K-ras LDR product to DNA array
The LDR reaction mixture is diluted with 20 μL of 2X hybridization buffer to a final buffer concentration of 300 mM MES, pH 6.0, 10 mM MgCl ₂ , 0.1% SDS, denatured at 94 ° C. for 3 minutes, and on ice Cooled by. The array was preincubated for 15 minutes at 25 ° C. in 1 × hybridization buffer. Coverwell (Grace, Sun River, Oreg.) Was attached to the array and filled with 30 μL of diluted LDR reaction mixture. The array was placed in a humidified culture tube and incubated in a rotary hybridization oven at 65 rpm for 1 hour at 20 rpm. Following hybridization, the arrays were washed in 300 mM bicine, pH 8.0, 10 mM MgCl ₂ , 0.1% SDS for 10 minutes at 25 ° C. The fluorescence signal was measured using a Scanarray 5000 (GSI Lumonics).

  LDR detection of seven specific K-ras mutations on a localizable universal microarray is shown in FIG. The three signals along the top right side of each array and those at the bottom are the criteria used for positioning. The following four addresses across the second column correspond to addresses # 1, # 2, # 3, and # 4 that are complementary to cZip1, cZip2, cZip3, and cZip4, respectively. Eight cell lines (ie, COL0205, LS180, SW1116, SW480, and DLD1) and tumor samples (G12S, G12R, and G12C) were accurately identified as mutations present. The wild type cell line COL0205 gives Cy3, Cy5 and fluorescein signals to address # 1. The wild type signal at address # 1 was used as a control for all tests. The LS180 cell line containing the Asp12 mutation gave a Cy3 signal at address # 2. The SW1116 cell line containing the Ala12 mutation gave a Cy5 signal at address # 2. The SW480 cell line containing the Val12 mutation gave a fluorescein signal at address # 2. G12S tumor samples containing the Ser12 mutation gave a Cy3 signal at address # 3. A G12R tumor sample containing the Arg12 mutation gave a Cy5 signal at address # 3. G12C tumor samples containing the Cys12 mutation gave a fluorescein signal at address # 3. The DLD1 cell line containing the Asp13 mutation gave a Cy3 signal at address # 4. The inaccurate signal seen in Zip4 of LS180 and SW1116 samples was due to sample contamination.

増幅は40ラウンドの94℃15秒間および60℃2分間の温度サイクル、続く65℃5分間の最終伸長段階により達成された。PCR後、1μLのプロテイナーゼK（18mg/mL）を添加し、反応液を70℃10分間加熱し、その後95℃で15分間反応を停止させた。予測された大きさの増幅産物の存在を確認するために各PCR産物1μLが3％アガロースゲル上で解析された。[Table 3] Primers designed for K-ras mutation detection by PCR / LDR / array hybridization

Amplification was achieved by 40 rounds of 94 ° C. for 15 seconds and 60 ° C. for 2 minutes, followed by a final extension step of 65 ° C. for 5 minutes. After PCR, 1 μL of proteinase K (18 mg / mL) was added, the reaction solution was heated at 70 ° C. for 10 minutes, and then the reaction was stopped at 95 ° C. for 15 minutes. 1 μL of each PCR product was analyzed on a 3% agarose gel to confirm the presence of the expected size amplification product.

LDR of K-ras DNA sample
The LDR reaction consists of 20 mM Tris HCl, pH 8.5, 5 mM MgCl ₂ , 100 mM KCl, 10 mM DTT, 1 mM NAD ⁺ , a total of 8 pmol LDR probe (4 fmol fluorescence labeled common probe added to each identification probe of 500 fmol), It was performed under paraffin oil in a 20 μL volume containing 1 pmol of PCR product from a cell line or tumor sample. Two probe mixtures were prepared, each containing either seven mutation-specific probes, three common probes and either a wild type discrimination probe for codon 12 or one for codon 13 (Table 3).

  The reaction mixture was preheated at 94 ° C. for 2 minutes, after which 25 fmol of wild type TthDNA ligase was added. The LDR reaction was cycled 20 rounds at 94 ° C for 15 seconds and 65 ° C for 4 minutes. A 2 μL aliquot of each reaction was mixed with 2 μL of gel loading buffer (8% blue dextran, 50 mM EDTA, pH 8.0-formamide (1: 5)), denatured at 94 ° C. for 2 minutes, and cooled on ice. 1 μL of each mixture was loaded onto a 10% denaturing polyacrylamide gel and electrophoresed at 1500 V on an ABI377 DNA sequencer.

Hybridization of K-ras LDR product to DNA array
The LDR reaction solution (17 μL) was diluted with 40 μL of 1.4 × hybridization buffer to a final buffer concentration of 300 mM MES, pH 6.0, 10 mM MgCl ₂ , 0.1% SDS. The array was preincubated for 15 minutes at 25 ° C. in 1 × hybridization buffer. Coverwell (Grace, Sun River, OR) was filled with 30 μL of diluted LDR reaction and allowed to attach to the array. The array was placed in a humidified culture tube and incubated at 65 ° C. for 1 hour and 20 rpm in a rotary hybridization oven. Following hybridization, the arrays were washed in 300 mM bicine, pH 8.0, 10 mM MgCl ₂ , 0.1% SDS for 10 minutes at 25 ° C. The fluorescence signal was measured using a microscope / CCD as described in the following paragraphs.

Imaging Arrays include a Molecular Dynamics FluorImager 595 (Sunnyvale, CA) or Princeton Instruments TE / CCD-512 TKBM1 camera (Trenton, NJ) Imaged using an Olympus AX70 epifluorescence microscope (Melville, NY). For analysis of fluorescently labeled probes on the fluoroimager, 488 nm excitation was used with a 530/30 emission filter. The spatial resolution of scanning is 100 μm per pixel. Results were analyzed using ImageQuaNT software provided with the instrument. Epi-illumination microscope is 100W mercury lamp, FITC filter cube (excitation 480/40, dichroic beam separator 505, emission 535/50), Texas red filter cube (excitation 560/55, dichroic beam separator 595, emission) 645/75) and 100mm macro objective lens. The macro objective lens allows an illustration of the field of view up to 15 mm in diameter and projects a 7 x 7 mm array area onto a 12.3 x 12.3 mm CCD matrix. Images were collected in 16-bit mode using Winview32 software provided with the camera. Analysis was performed using Scion images (Skion, Fredrick, MD).

Example 2 Amplification of BRCA1 and BRCA2 exons for wild-type and mutant allele PCR / PCR / LDR detection Ligase detection reaction (“LDR”) (Khanna, M et al., “Primary colorectal tumors, incorporated herein by reference) Multiple PCR / LDR for Detection of K-ras Mutations in Primary Colon Tumors (Oncogene, 18: 27-38 (1999)) followed by equal amplicons Modified PCR (PCR / PCR) that amplifies to (incorporated herein by reference, Belgrader et al., “A Mulitplex PCR-Ligase Detection Reaction Assay for Human Identity Testing), Genome Science and Technology 1: 77-87 (1996)) was used to detect small insertions and deletions. Figure 20 shows how appropriate exon multiplex amplification is performed to ensure that all products are amplified evenly: a limited number of PCR cycles are performed using gene-specific primers. And several additional rounds of amplification priming from the universal sequence located at the 5 'end of the PCR primer. This method minimizes amplification bias based on primer-specific effects. LDR is then used to detect both wild type and mutant versions of the query sequence. Ligated oligonucleotide probes hybridize to both wild type and mutant products, but are ligated only when both probes are perfectly matched without gaps or overlaps. Products can be either electrophoretically separated or hybridized to a microarray for identification.

PCR was performed as a single tube multiplex reaction to simultaneously amplify BRCA1 exon 2 and exon 20 and BRCA2 exon 11. Genomic DNA was extracted from blood samples of Ashkenazi Jewish individuals and supplemented with 100 ng DNA, 400 μM of each dNTP, 4 mM MgCl ₂ (10 mM Tris HCl, pH 8 at 25 ° C.). (3, 50 mM KCl), 1 U Amplitude Gold and amplified in a 25 μL reaction mixture with 2 pmol of each gene specific primer containing either universal primer A or B at the 5 ′ end. Table 4 shows primers and probes for detection of BRCA1 and BRCA2 mutations using PCR / LDR / array hybridization as follows.

[Table 4]
Primers and probes designed for BRCA1 and BRCA2 mutation detection by PCR / LDR / array hybridization

The reaction was overlaid with mineral oil and preincubated at 95 ° C. for 10 minutes. Amplification was performed for 15 cycles as follows: 94 ° C for 15 seconds, 65 ° C for 1 minute. A 25 μL aliquot of the second reaction mixture containing each universal primer A and B was added through 25 pmol of mineral oil. The cycle was repeated using a 55 ° C. annealing temperature. The reaction was then digested for 10 minutes at 55 ° C. with 2 μL of 1 mg / ml proteinase K / 50 mM EDTA. Proteinase K was erased by a final incubation at 90 ° C. for 15 minutes. For LDR, oligonucleotide synthesis and purification is as previously described (Khanna, M et al., “Multiple PCR / LDR for detection of K-ras mutations in primary colon tumors, incorporated herein by reference. (Multiplex PCR / LDR for Detection of K-ras Mutations in Primary Colon Tumors) "Oncogene 18: 27-38 (1999)). Tth DNA ligase was overproduced and purified as described separately (Luo et al., “Identification of Essential Residues in Thermus Thermophilus, incorporated herein by reference. “Nucleic Acids Research 24: 3079-3085 (1996)” and Barany et al. “Cloning, Overexprescion, and Nucleotide Sequence of a Thermostable DNA Ligase Gene” Gene 109: 1-11 (1991)). LDR was performed in a 20 μL reaction containing 500 fmol of each probe, 2 μL of amplified DNA and 20 mM Tris HCl, pH 7.6, 10 mM MgCl ₂ , 100 mM KCl, 10 mM DTT, 1 mM NAD ⁺ . The reaction was heated to 94 ° C. for 1.5 minutes before addition of 25 fmol of Tth DNA ligase and then subjected to 20 cycles at 94 ° C. for 15 seconds and 65 ° C. for 4 minutes (see Table 4).

  Three BRCA1 and BRCA2 founder mutations (BRCA1 185delAG, BRCA1 5382insC, BRCA2 6174delT) from the Ashkenazi Jewish population as model systems (Rahman et al., “Genetics of Breast Cancer Susceptibility, incorporated herein by reference. (The Genetics of Breast Cancer Susceptibility), Annu. Rev. Genet. 32: 95-121 (1998)), the assay facilitates these mutations in multiplexed reactions in more than 80 DNA samples. Detected. FIG. 21A shows a representative LDR gel that detects three BRCA mutations. Adding different length “tails” to the LDR oligonucleotide probe by fluorescently end-labeling the discriminating upstream oligonucleotide with either FAM (relative to the wild type) or TET (for mutants) The ligation products were easily identified based on the label and mobility on the polyacrylamide gel. Wild-type products are identified on the right side of the gel. Mutant products are identified at the top of the gel. Electrophoretic separation was performed at 1400 V using an 8M urea-10% polyacrylamide gel and an ABI373 DNA sequencer. Fluorescent ligation products were analyzed and quantified using ABI GeneScan 672 software.

  The analysis was subsequently extended to detect mutations in DNA pooled samples. DNA with known mutations was diluted 1: 2, 1: 5, 1:10 and 1:20 with wild-type DNA prior to PCR amplification and then subjected to LDR. These simulations show that PCR / PCR / LDR can successfully detect the presence of all three mutations when known mutant DNA is diluted 1:20 using wild-type DNA prior to amplification. It showed that. FIG. 21B shows BRCA1 and BRCA2 mutation detection in pooled DNA samples. DNA with known mutations was diluted in wild type DNA prior to amplification. Multiple LDR ligation products are shown for each dilution. BRCA1delAG, BRCA1insC, and BRCA2delT mean that there is only one mutation. Multiplexed LDRs directed only to mutant sequences use 500 fmol of each LDR oligonucleotide probe. Three mutations means that there are all three mutations. Multiple LDRs directed to mutant sequences only use 500 fmol of each LDR oligonucleotide. 1:20 3 mutations + wild type control means that all 3 mutations are present. Multiplexed LDRs are directed to both mutant and wild type sequences using 500 fmol and 25 fmol of each LDR oligonucleotide probe, respectively. The pooled experiment was repeated using 249 blinded Ashkenazi Jewish DNA samples. The tubes containing the blinded DNA are arranged in a 9x9 grid format, and aliquots from each tube span the rows and then the columns to produce the combined DNA for each row and each column in one tube. Integrated down. This was done to uniquely classify each sample using intersections in a grid format. The pooled DNA was then subjected to amplification and LDR as described above. Of the 249 samples, 248 were correctly typed. One sample that was incorrectly identified as the wild type proved to be too dilute and fell below the detection limit when mixed with the other nine samples with higher concentrations. The number of individual reactions performed was reduced from 249 to 96 by this strategy (55 pool samples and 41 individual samples used for validation).

In addition to gel-based detection, mutation identification was also achieved by screening reaction products using universal DNA microarrays (Gerry et al., “Multiple of small point mutations, incorporated herein by reference. Universal DNA Microarray Method for Multiplex Detection of Low Abundance Point Mutations "J. Mol. Biol. 292: 251-262 (1999)). The microarray was processed and spotted as previously described using a Pixis 5500 robot enclosed in a humidified chamber (Cartesian Technology, Irvine, CA) (Gerry et al., Incorporated herein by reference). "Universal DNA microarray method for multiplex detection of small amount of point mutations" J. Mol. Biol. 292: 251-262 (1999)). Briefly, the LDR reaction was hybridized in 32 μL containing 300 mM MES, pH 6.0, 10 mM MgCl ₂ , 0.1% SDS in a rotating chamber at 65 ° C. for 1 hour. Olympus Provis AX70 using a 100 W mercury burner, Texas Red Filter Cube, and Princeton Instruments TEK512 / CCD camera after washing in 300 mM bicine, pH 8.0, 10 mM MgCl ₂ , 0.1% SDS for 10 minutes at 25 ° C. Imaged on a microscope. 16-bit grayscale images were captured using a MetaMorph imaging system (Universal Imaging Corporation, Westchester, PA) and rescaled to narrower the LDR signal before converting to 8-bit grayscale . The 8-bit image was colorized using Adobe Photoshop to give the Cy3 signal red.

  Preliminary microarray experiments using probes designed in a gel-based format (ie, differentially labeled discriminating probes and the same common probe) facilitated the wild-type and mutant versions of the three BRCA sequences on the array (See WO97 / 31256 to Barany et al., Incorporated herein by reference). In this edition, both sequence types were directed to the same address (eg, BRCA1 185delAG and BRCA1 185 wild type were both directed to Zip code 1). Although this format has proven successful, PCR / PCR / LDR has the ability to detect hundreds of mutations in a single tube reaction, and this design is for such large-scale mutation identification experiments. Is not the best application of the array. In order to establish an experimental paradigm for future studies, the localizable format was expanded by selecting sequences within each amplicon using the LDR ligation product as a control. Thus, rather than having to detect the wild-type sequence for each mutant LDR product, this format uses a single product that serves as a positive control for many different sequence variants within an amplicon ( (See Figure 22). One advantage of this format is to minimize oligonucleotide synthesis. In addition, the use of each of the 64 sites is maximized. Since the number of LDR ligation products detectable at a single address is limited by the number of fluorescent labels separated in the currently available spectrum, limiting the control to a particular area of the array Allows detection of one more sequence variant at each address. In the experiments described below, the control and mutant LDR ligation products for the introduced sites were directed to 6 separate addresses on a 64 site array.

  All three frameshift mutations were detectable by hybridization to a universal array (Figure 23). FIG. 23A shows that each of the control and variant sequences are placed at specific addresses on the array surface. Control signals were directed to the top three addresses. Mutant signals were directed to the bottom three. FIG. 23B shows the signal generated by wild type DNA. Figures 23C, E and G show representative hybridization of individual DNA samples. FIGS. 23D, F and H show representative hybridization of each mutation using pooled DNA samples from Ashkenazi individuals. Mutations are identified on the far right.

  Only six possible address combinations became visible following hybridization, and no additional signal was detected at any other unused addresses. Thus, Zip code hybridization is very specific. Control and mutant signals were clearly present for each mutation derived from a DNA sample from a single individual (FIGS. 23C, E and G). The pooled DNA used in analyzing the 249 DNA samples above produced the same mutated signal as found in the gel-based assay (FIGS. 23D, F and H). In each case, the array reproduced the gel results.

  These results demonstrate that universal microarray analysis of PCR / PCR / LDR products allows for the rapid identification of small insertion and deletion mutations in both clinical diagnostic and population study situations.

Example 3 p53 chip hybridization and wash conditions To determine which method can produce minimal background noise without significant loss of signal, three parameters (presence or absence of non-specific competitive DNA, temperature , And wash buffer composition) were changed in different combinations. Hybridization was performed at 100 μg / ml sheared salmon sperm DNA (FIGS. 16B, D, J and L), 250 μg / ml sheared salmon sperm DNA (FIGS. 16F, H, N and P), or non-specific competitor DNA (FIG. 16A). , C, E, G, I, K, M and O). Washing was performed for 10 minutes using 4 different conditions. Standard wash buffer (300 mM _{bicine, pH8.0,10mM MgCl 2, 0.1% SDS} ) at room temperature using a (FIG. 16A, B, E and F). Room temperature (Figure 16I, J, M and N) using standard wash buffer supplemented with 10% formamide. 50 ° C. using standard wash buffer (FIGS. 16C, D, G and H) or 50 ° C. using standard wash buffer supplemented with 10% formamide (FIGS. 16K, L, O and P). The number in the upper right of each panel indicates the pixel density of p53 exon 5 control (Zip code 1) for each condition. The loss percentage shown on the right side of the number compares the left and right panels directly adjacent to the calculated percentage. The references (Cy3, Cy5 and fluorescein) are spotted horizontally in the upper left of the chip and vertically in the lower right area to give direction. Zip code 1 is located just below the reference in the upper left area. Subsequent Zip codes are spotted in numerical order from left to right.

PCR was performed as a single tube multiplex reaction to amplify p53 exons 5 and 7 simultaneously. Commercially available genomic DNA derived from leukocytes is 100 ng DNA, 400 μM of each dNTP, 1 × PCR buffer II supplemented with 4 mM MgCl ₂ (10 mM Tris HCl, pH 8.3 at 25 ° C., 50 mM KCl), 1 U AmpliTac Gold And amplified in a 25 μL reaction mixture containing 2 pmol of each gene specific primer with either universal primer A or B at the 5 ′ end (see Table 5). The reaction solution was overlaid with mineral oil and preheated at 95 ° C. for 10 minutes. Amplification was performed for 15 cycles: 94 ° C for 15 seconds, 65 ° C for 1 minute. A 25 μL aliquot of the second reaction mixture containing 25 pmol of each universal primer A and B was added through mineral oil. The cycle was repeated using a 55 ° C. annealing temperature. The reaction was then digested for 10 minutes at 55 ° C. with 2 μL of 1 mg / ml proteinase K / 50 mM EDTA. Proteinase K was erased by a final incubation at 90 ° C. for 15 minutes. For LDR, oligonucleotide synthesis and purification is as previously described (Khanna, M et al., “Multiple PCR / LDR for detection of K-ras mutations in primary colon tumors, incorporated herein by reference. (Multiplex PCR / LDR for Detection of K-ras Mutations in Primary Colon Tumors) "Oncogene 18: 27-38 (1999)). Tth DNA ligase was overproduced and purified as described separately (Luo et al., “Identification of Essential Residues in Thermus Thermophilus, incorporated herein by reference. DNA Ligase), Nucleic Acids Research 24: 3079-3085 (1996) and Barany et al. Cloning, Overexpression, and Nucleotide Sequence of a Thermostable DNA Ligase Gene Gene 109: 1-11 (1991)). LDR was performed in a 20 μL reaction containing 500 fmol of each probe, 2 μL of amplified DNA and 20 mM Tris HCl, pH 7.6, 10 mM MgCl ₂ , 100 mM KCl, 10 mM DTT, 1 mM NAD ⁺ . The reaction was heated at 94 ° C. for 1.5 minutes and 25 fmol of Tth DNA ligase was added, followed by 20 cycles of 94 ° C. for 15 seconds and 65 ° C. for 4 minutes. See Table 5 showing oligonucleotide primers and probes designed for detection of p53 mutations by PCR / LDR / array hybridization as follows.

[Table 5]
Primers and probes designed for p53 mutation detection by PCR / LDR / array hybridization

  PCR primers are specifically designed to amplify regions within and around the p53 gene. After 15 rounds of amplification at a high Tm (ie 65 ° C.) with longer gene-specific primers (1-2 pmol for each reaction), two universal primers (bold capital letters) were added at 50 pmol in 50 μL, And the product is further cycled to 20 rounds of amplification.

  Allele-specific LDR probes contained a fluorescent label (Fam, Tet, Cy3 or Cy5) at the 5 ′ end and a discriminating base at the 3 ′ end. A non-genomic sequence was added to the 5 'end of some probes (designated in bold) to control the size of the final ligation product for gel-based assays. The common LDR probe contained a 5 'phosphate group (p) and a C-3 blocking group (B) at its 3' end. A common LDR probe used in array-based detection has a Zip coding sequence at its 3 'end (designated in lower case).

LDR reaction was hybridized in 32 μL with 300 mM MES, pH 6.0, 10 mM MgCl ₂ , 0.1% SDS with or without 100 μg / ml sheared salmon sperm DNA at 65 ° C. for 1 hour in a rotating chamber . 100 W mercury burner, Texas Red Filter Cube, and Princeton after washing in 300 mM bicine, pH 8.0, 10 mM MgCl ₂ , 0.1% SDS at 25 ° C. for 10 minutes or 50 ° C. for 10 minutes with or without 10% formamide It was imaged on an Olympus Provis AX70 microscope using an instrument TEK512 / CCD camera. The 16-bit grayscale image was captured using a metamorph imaging system (Universal Imaging Corporation) and rescaled to narrow the LDR signal more narrowly before conversion to 8-bit grayscale. This 8-bit image was inverted using Adobe Photoshop to give the Cy3 signal black.

Example 4 p53 chip hybridization indicating the presence of mutations in colorectal tumor-derived DNA The p53 chip is capable of detecting the presence of 75 different mutations present in exons 5, 6, 7 and 8. 144 LDR oligonucleotides are used (see Table 5). FIG. 24 is an example of p53 mutation detection based on a microarray using colon tumor-derived DNA. The status of the mutation in each sample and the Zip code expected to capture the signal are shown to the right of each panel. The figure shows the Cy3 background due to contaminating fluorescence in the spotted Zip code in the bottom right panel (Figure 24H). In order to amplify p53 exons 5, 6, 7 and 8 simultaneously, PCR was performed as a single tube multiplex reaction. Genomic DNA extracted from colorectal tumors is 100 ng DNA, 400 μM of each dNTP, 1 × PCR buffer II supplemented with 4 mM MgCl ₂ (10 mM Tris HCl, pH 8.3 at 25 ° C., 50 mM KCl), 1 U ampli tack Amplified in a 25 μL reaction mixture containing gold and 2 pmol of each gene specific primer with either universal primer A or B at the 5 ′ end (see Table 5). The reaction solution was overlaid with mineral oil and pre-heated at 95 ° C. for 10 minutes. Amplification was performed for 15 cycles as follows: 94 ° C for 15 seconds, 65 ° C for 1 minute. A 25 uL aliquot of a second reaction mixture containing 25 pmol of each universal primer A and B was added through mineral oil. The cycle was repeated using a 55 ° C. annealing temperature. The reaction was then digested for 10 minutes at 55 ° C. with 2 μL of 1 mg / ml proteinase K / 50 mM EDTA. Proteinase K was erased by a final incubation at 90 ° C. for 15 minutes. For LDR, oligonucleotide synthesis and purification is as previously described (Khanna, M et al., “Multiple PCR / LDR for detection of K-ras mutations in primary colon tumors, incorporated herein by reference. (Multiplex PCR / LDR for Detection of K-ras Mutations in Primary Colon Tumors) "Oncogene 18: 27-38 (1999)). Tth DNA ligase was overproduced and purified as described separately (Luo et al., “Identification of Essential Residues in Thermus Thermophilus, incorporated herein by reference. DNA Ligase), Nucleic Acids Research 24: 3079-3085 (1996) and Barany et al. Cloning, Overexpression, and Nucleotide Sequence of a Thermostable DNA Ligase Gene Gene 109: 1-11 (1991)). LDR was performed in a 20 μL reaction containing 500 fmol of each probe, 2 μL of amplified DNA and 20 mM Tris HCl, pH 7.6, 10 mM MgCl ₂ , 100 mM KCl, 10 mM DTT, 1 mM NAD ⁺ . Two reactions were performed for each sample containing LDR probes designed to hybridize to the upper or lower strand of the p53 sequence. The reaction was heated at 94 ° C. for 1.5 minutes prior to the addition of 25 fmol Tth DNA ligase and then subjected to 20 cycles at 94 ° C. for 15 seconds and 65 ° C. for 4 minutes (see Table 5). The LDR reaction was hybridized in 32 μL containing 300 mM MES, pH 6.0, 10 mM MgCl ₂ , 0.1% SDS containing 100 μg / ml sheared salmon sperm DNA at 65 ° C. for 1 hour in a rotating chamber. Olympus Provis AX70 using a 100W mercury burner, Texas Red filter cube, and Princeton Instruments TEK512 / CCD camera after washing in 300 mM bicine, pH 8.0, 10 mM MgCl ₂ , 0.1% SDS for 10 minutes at 50 ° C. Imaged on a microscope. The 16-bit grayscale image was captured using a metamorph imaging system (Universal Imaging Corporation) and rescaled to narrow the LDR signal more narrowly before conversion to 8-bit grayscale. The 8-bit image was first inverted using Adobe Photoshop to give the Cy3 signal black, and then an image of each sample derived from hybridization using LDR targeting the upper or lower strand of the p53 sequence. Overlaid and overlaid. The result of this process is shown in FIG.

Example 5 Optimized Zip Coding Sequence Construction Using Tetramer Universal DNA array is a concept that uses diffuse sequences to uniquely tag LDR products so that each is captured at a unique site. Designed based on. The heart of this concept is a design of 36 tetramers, each differing by at least 2 bases from each other (see Table 6).

これらの36個の四量体を6個のセットに組み合わせることにより、24塩基長のアドレスを構築することが可能である。

By combining these 36 tetramers into 6 sets, it is possible to construct a 24 base length address.

  The 1296 array can be designed based on the concept of alternate tiling of a given set of tetramers. These capture oligonucleotides differ from their neighbors in three alternating sites, but are the same in the other three sites, ie (first = A, third = C and fifth = E sites). Thus, each capture oligonucleotide differed from any other one by at least 6 out of 24 sites. Furthermore, these differences were distributed over the length of the capture oligonucleotide. When the correct capture oligonucleotide was placed at the wrong address, the Tm difference was expected to be above 24 ° C. Nevertheless, one possibility of this design type is that three adjacent tetramers (ie ABC) in a given set of sites match another capture oligonucleotide, but a different set of sites ( That is, in BCD).

  Since the optimal surface is a three-dimensional porous surface, there is a great opportunity for a given LDR product to be captured at the correct address. Even if the LDR product is transiently dissociated from a given oligonucleotide in the correct address, another oligonucleotide in the same address is quickly found and hybridized. In preliminary studies, changes expected to change Tm (ie use of propynyl derivatives) did not significantly affect the yield of correctly hybridized products. Thus, hybridization can be kinetically controllable. To minimize the possibility of even a low level of alternating hybridization between two well-related oligonucleotides, sequence to maximize the difference between the order of the tetramers with a 24mer capture oligonucleotide Can be designed.

  An overview for designing such an arrangement is as follows.

1. Create three rows of 3 sets of 36 tetramers, including a total of 46,656 permutations (= 36 × 36 × 36).

2. Computer-calculate 46,656 12mer Tm using the Molecular 6.0 Biology Insights (Cascade, CO) Oligo 6.0 program and sort the list according to the predicted Tm value.

3. Remove 12mer (tetramer # 1 to # 7) containing 1 GC base in each tetramer or 12mer (tetramer # 28 to # 36) containing 3 GC bases in each tetramer. This process removes the extreme value of the Tm value. Remove 12mer with Tm value less than 24 ℃. Remove the remaining 12mer (ie 9-9-9) with 3 repeated tetramers.

4. Each time Tm rises twice, a 12-mer set is grouped by Tm as a new group. The value was determined by dividing Tm by 2 and rounding down to an integer.

5. Randomize the list and split it into odd and even 12mers. Invert the second list and add it to the end of the first list to form 6 tetramers = 12,880 address candidates. The sequences are ligated to determine the 24mer Tm value.

6. Select only 6 tetramers with Tm values between 75 and 84. Regenerate unused trimers, create 6 new tetramers with increased Tm, and add them back to the list.

7. Cut down the list using 13 selection conditions as listed in Table 7. “1” indicates that there is a match at the site, and “0” indicates that there is no match. Whenever two candidate addresses match in one of the conditions, it was removed from the candidate list and returned to the unused trimer list.

8. The above 13 conditions reduced the list to 9,650 address candidates.

9. The 13 conditions above remove 4 consecutive matches and remove 3 consecutive matches that are in the same arrangement as another one. However, sequences that are similar but converted by one or two tetramer units are not removed. To eliminate these types of artifacts, they were copied under the original six tetramers and corrected with one or two tetramers as shown in Figure 8 below.

10. These three selections screened a list of 8,894 candidate capture oligonucleotides, 4,798 more than 4,096 targets in a 64 × 64 address array. These capture oligonucleotide sequences have the following characteristics: (1) There are no examples of four consecutive tetramers that are identical, either when the capture oligonucleotide sequences are placed together, or corrected or split from each other. (2) There is no example of three tetramers that are identical in a row when the capture oligonucleotide sequences are placed from one end to each other. (3) There is no example in which 4 tetramers out of 6 tetramers are in the same site.

11. For further selection, three consecutive corrected sequences were deleted as shown in Table 9.

12. These 6 selections screened from a list of 3,038 candidate capture oligonucleotides, more than 2,500 more selective targets in a 50 × 50 address array. These capture oligonucleotide sequences have the following characteristics: (1) There is no example of four consecutive tetramers that are identical either when the capture oligonucleotide sequences are placed together or corrected for each other. (2) There is no example of three consecutive tetramers that are identical when the capture oligonucleotide sequences are either positioned from one end to each other or corrected for each other. (3) There is no example in which 4 tetramers out of 6 tetramers are in the same site.

13. The order of the tetramer was reversed and a new Tm value was calculated and added back to (6,076), after which excess was removed as described in steps 7-11 above. The list of candidates increased slightly to 3,270. Therefore, a method for concentrating unused trimers was needed.

14. A list of trimers used at all sites was used to determine the available (unused) trimers and build a new set of 6 tetramers. In order to increase the proportion of 6 tetramers with Tm values in the range of 75 ° C to 84 ° C, trimers with predicted Tm values of 34 ° C to 50 ° C were inverted and used together (ie Tm34's Trimer ABC fused to Tm50 trimer DEF, Tm38 trimer ABC fused to Tm46 trimer DEF, etc.).

15. A set of 6 tetramers was constructed and the trimers created at the junction (ie, sites BCD and CDE) were retested against the used trimer list and they did not match Six tetramers were reused. The six tetramers that were not those discrepancies were added to the 3,270 candidate list, reordered, and the extras removed as described in steps 7-11. The candidate list has been expanded to 4,035.

16. This process was repeated two more times to create a final list of 4,633 capture oligonucleotides, 537 sequences more than 4,096 targets in the 64x64 address array (see tetramers in Table 6). Figure 25). These capture oligonucleotide sequences have the following characteristics: (1) There is no example of four consecutive tetramers that are identical, either when the capture oligonucleotide sequences are placed together or corrected for each other. (2) There is no example of three consecutive tetramers that are identical, either when the capture oligonucleotide sequences are positioned from one end to each other or when corrected for each other. (3) There is no example in which 4 tetramers out of 6 tetramers are in the same site.

17. Using a list of 4,633 capture oligonucleotides, smaller for 8 × 8 = 64 address array, 8 × 12 = 96 address array, 16 × 24 = 384 address array, and 20 × 20 = 400 address array A list has been created. As selection criteria, capture oligonucleotides that share a common tetramer pair were selectively removed from the list. By listing all dimer pairs that were identical at a given site (ie, AB = AB), the list was reduced to 465 capture oligonucleotide sequences (FIG. 26 with reference to tetramers in Table 6). By a second round of dimer pairs that are similar in adjacent sites (ie AB = BC, BC = CD, etc.) and by removing all dimer pairs that have been used more than twice, the set Reduced to 96 capture oligonucleotide sequences (Figure 27 with reference to tetramer in Table 6). Finally, a list of 65 capture oligonucleotide sequences was generated by ensuring that no dimer was used more than once (Figure 28 with reference to tetramer in Table 6).

18. As shown in FIG. 29 (see tetramer in Table 6), which includes a list of 4,633 such capture oligonucleotides, the capture oligonucleotides are also PNAs (ie, peptide nucleotide analogs). It can also be. PNA capture oligonucleotides are of the 20mer unit type. PNA offers the advantage of increasing the Tm of an oligonucleotide from 1.0 ° C to 1.5 ° C on average by 1 base. Therefore, the Tm values of the oligomers listed in FIG. 29 are considered to be 20 ° C. (or more) higher on average when synthesized as PNA type. Therefore, it is considered that the address need only be 20 mer or less in the PNA type. These sequences are amenable to faster synthesis by considering two alternative methods. In the first method, the 36 tetramers listed in Table 6 are first synthesized, and then the 5 tetramers are concatenated in the correct order to form the sequence listed in FIG. The Alternatively, the PNA oligomer may be synthesized using a lithographic synthesis method. In standard lithographic synthesis, 4 bases, ie ACGT in the first site, ACGT in the second site, etc. are used over and over, and 4 × 20 = 80 masking is considered necessary. . The current sequence listed in FIG. 29 is easier to adapt to synthesis by masking, 62 times or even less by changing the masking top number. 62 maskings are thought to allow the addition of PNA monomers in the following order:

であれば、ちょうど20回のマスキングを用いて終了することが可能と考えられる。異なる配列はより多くのマスキングを必要とすると考えられる。したがって、20merは異なる数のマスキングを用いて終了する。62回未満のマスキングを必要とする5個の異なるアドレスZipID#1、2、3、4および26の合成のための例が以下に提供される。これらの例では、正しい配列を達成するために、マスキングの使用はその塩基に下線を引くことで指示される。

これにより、20merを合成するための標準的アプローチに必要とされる80マスクよりもずっと少なく、それどころか16merの標準PNAの作製に必要な64マスクよりも少ない、62マスク以下を用いる平板アプローチを用いて、20merのPNAアドレスが合成されうる。For a given sequence, the masking that allows the sequence at the next base is released at that address. As an example, if the array is

If so, it would be possible to finish using exactly 20 maskings. Different sequences are thought to require more masking. Thus, 20mer ends with a different number of maskings. Examples for the synthesis of 5 different addresses ZipID # 1, 2, 3, 4 and 26 that require less than 62 maskings are provided below. In these examples, the use of masking is indicated by underlining the base to achieve the correct sequence.

This uses a flat plate approach that uses 62 masks or less, much less than the 80 masks required for the standard approach to synthesize 20 mer, and even less than the 64 masks required to create a standard 16 mer PNA. , 20mer PNA addresses can be synthesized.

  All addresses were selected to have Tm values between 75-84 ° C. The distribution of Tm values is more or less independent of the number of capture oligonucleotides. In general, a simple Tm = 4 (G + C) +2 (A + T) rule is up to 10 ° C in many cases, while addresses with high G + C content gave higher Tm values Go crazy. Values for 96, 465, and 4,633 capture oligonucleotides are shown in FIGS. 30, 31, and 32, respectively. The sorted Tm values for the 4,633 list of capture oligonucleotide probes are shown in FIG. The slope of the Tm value is relatively flat and most capture oligonucleotides (80%) have Tm values from 75 ° C to 80 ° C including both ends. FIG. 34 shows the utilization of tetramers in a list of 65, 96, 465 and 4633 capture oligonucleotides made in accordance with the present invention. If the tetramer distribution is completely random, each tetramer should be presented at a rate of 2.7%. However, the selection is biased towards higher Tm capture oligonucleotide probes. Thus, tetramers that tend to decrease Tm values, ie # 7 = TACA, are underpresented, while tetramers that tend to increase Tm values, ie # 29 = TGCG, are overpresented.

  This method of mutation detection has three orthogonal components. (1) first PCR amplification, (2) liquid phase LDR detection, and (3) solid phase hybridization capture. Thus, the background signal from each stage can be minimized, and as a result, the overall sensitivity and accuracy of the method is significantly enhanced relative to those provided by other strategies. For example, the “sequencing by hybridization” method limits throughput, (1) multiple rounds of PCR or PCR / T7 transcription, (2) processes PCR amplification products to make them fragmented or single stranded And (3) long hybridization times (10 hours or longer), Guo et al., Nucleic Acids Research 22: 5456-5465 (1994), Hacia et al., Incorporated herein by reference. Nat. Genet. 14441-447 (1996), Chee et al., Science 274: 610-614 (1996), Cronin et al., Human Mutation 7: 244-255 (1996), Wang et al., Science 280: 1077-1082 (1998). Schena et al., Proc. Natl. Acd. Sci. USA 93: 10614-10619 (1996) and Shalon et al., Genome Res. 6: 639-645 (1996)). In addition, since the immobilized probes on these arrays have a wide range of Tm, it is necessary to perform the hybridization at a temperature of 0 ° C. to 44 ° C. The result is increased background noise and spurious signals due to mismatch hybridization and non-specific binding, for example for small insertions and deletions in repetitive sequences (Hacia et al., Nat. Genet. 14441-447 (1996), Cronin et al., Human Mutation 7: 244-255 (1996), Wang et al., Science 280: 1077-1082 (1998), and Southern EM, Trends in Genet, 12: 110-115 ( 1996)). In contrast, the method of the present invention allows for multiplex PCR in a single reaction (Belgrader et al., Genome Sci. Technol. 1: 77-87 (1996), incorporated herein by reference), and the product is single stranded. All point mutations, including shifts in repetitive sequences, can be easily identified without the need for additional steps to convert to type (Day et al., Genomics, 29: incorporated herein by reference). 152-162 (1995)). Another DNA array is subject to different hybridization efficiencies due to sequence variations or the amount of target present in the sample. By using this method of designing diffuse address sequences with similar thermodynamic properties, hybridization can be performed at 65 ° C, resulting in more stringent and rapid hybridization. Uncoupling the mutation detection step from the hybridization step provides an aim for quantification of LDR products that has already been achieved using gel-based LDR detection.

  Arrays spotted on multimeric surfaces provide a significant improvement in signal capture compared to arrays spotted or directly in situ synthesized on glass surfaces (Drobyshev et al., Gene, incorporated herein by reference). 188: 45-52 (1997), Yershov et al., Proc. Natl. Acd. Sci. USA 94: 4913-4918 (1996) and Parinov et al., Nucleic Acids Res. 24: 2998-3004 (1996)). However, the multimeric surface of the present invention allows 24mer capture oligonucleotides to easily penetrate and covalently bind, while multimers described by others are limited to using 8 to 10mer addresses. Has been. It was further found that LDR products 60 to 75 nucleotide bases in length also penetrate and subsequently hybridize to the correct address. As an added benefit, the multimer produces little or no background fluorescence and does not show non-specific binding of the fluorescently labeled oligonucleotide. Finally, spot oligonucleotides and covalently attached capture oligonucleotides at distinct addresses do not “bleed over” to adjacent spots, eliminating the need to physically isolate the site, for example by cutting a gel pad .

  The present invention relates to a strategy for high-throughput mutation detection that differs significantly from other array-based detection systems previously presented in the literature. Coordinated with polymerase chain reaction / ligase detection reaction (PCR / LDR) assay performed in solution, present as 50% of genetic and its gene sequences, or less than 1% of sporadic and wild type sequences The arrays of the present invention allow for the accurate detection of single base mutations. This sensitivity is achieved because the diffusely localizable array-specific portion of the LDR probe directs each LDR product to a specified address on the DNA array, while thermostable DNA ligase provides the specificity of mutation discrimination Is done. The addressable array of the present invention is universal because the address sequence remains constant and the complement can be added to any LDR probe set. Thus, a single array design can be programmed to detect a wide range of genetic variations.

  A robust method for rapid mutation detection at large potential sites of a large number of genes strongly promises improved diagnosis and treatment of cancer patients. Non-invasive testing for mutation analysis of saliva, sputum, urine and fecal shed cells significantly simplifies and improves surveillance in high-risk populations and reduces the cost and discomfort of endoscopy It is considered possible to lead to more effective cancer diagnosis at an early and treatable stage. The feasibility of detecting a drop-off mutation has been clearly demonstrated in patients with known and genetically characterized tumors (Sidransky et al., Science, 256: 102-105, incorporated herein by reference). 1992), Nollau et al., Int. J. Cancer, 66: 332-336 (1996), Calas et al., Cancer Research, 54: 3568-73 (1994), Hasegawa et al., Oncogene 10: 1413-16 (1995) and Wu. Et al., “Early Detection of Cancer Molecular Marker” (edited by Lippman et al.) (1994), effective screening before symptoms appear, with a myriad of infrequent potential mutations being minimal Would need to be identified with false positive and false negative signals. In addition, the integration of techniques for determining genetic changes in tumors with clinical information about the likelihood of a therapeutic response will fundamentally change how patients with more advanced tumors are selected for treatment. Yes. Many candidate genes need to be tested in large clinical trials for the identification and confirmation of reliable genetic markers. High-cost microfabricated chips can be made with 100,000 addresses, but none of them are infrequently required to accurately score mutation profiles in such clinical trials. The ability to identify mutations has not been demonstrated (Hacia et al., Nat. Genet. 14441-447 (1996), Chee et al., Science 274: 610-614 (1996), Kozal et al., Incorporated herein by reference. Nat. Med. 2: 753-759 (1996) and Wang et al., Science 280: 1077-1082 (1998) The universal positionable array method of the present invention eliminates a large number of genes associated with tumor progression. Loss and amplification quantification and the ability to quickly and reliably identify low frequency mutations at multiple codons in a myriad of genes.In addition, for mRNA expression profiles, LDR universal arrays are K-, N- And very similar like H-ras In addition, as new therapies targeting specific genes or specific mutant proteins are developed, the importance of rapid and accurate high-throughput genetic testing is undoubtedly important It is thought to increase.

Example 6: Computer software for designing a positionable array that avoids binding to the target sequence In designing a positionable array, ensuring that the target sequence does not hybridize to the capture probes on the array is important. A computer program was designed for this purpose, as described below. The program places a series of sequences that match any array sequence in Nx N adjacent nucleotide sites. Parameters x and N are set by the user.

大文字の対立遺伝子が実際に一致した部位を表し、小文字の対立遺伝子が許容されるミスマッチを示しており、括弧内の領域は同定された最長の一致を表す。The program delivers output to screens and files. The screen output summarizes the number of sequence comparisons where the longest match is i of the M bases, M is greater than or equal to N, and i is greater than or equal to Mx. The output file gives an overview of the information provided on the screen and shows the actual matches for each sequence set. An example of file output is shown below.

Uppercase alleles represent the actual matched sites, lowercase alleles indicate an acceptable mismatch, and the region in parentheses represents the longest identified match.

  The program is written in ANSI C for the purpose of portability between platforms. The exact software used is described in FIG. The program accepts a straight text input file. The sequence may include a standard ambiguity code (eg, code Y corresponds to either C or T).

  Each of the F1 sequences in the input file # 1 is compared with each of the F2 sequences in the input file # 2, resulting in a total F1 × F2 comparison. For each set, the two sequences are compared at N consecutive sites at a time for all possible configurations. If at least Nx matches of N adjacent sites are detected, the compared sites are incremented by 1 (ie, after i increases, the match criterion is N-x + of N + i sites). i). This process is repeated until no match is found that meets the match criteria. The longest match of a sequence pair is defined as the match containing the longest value of N + i as opposed to the longest value of N-x + i. Therefore, the user should clearly repeat all analyzes with different levels of stringency (ie, x = 0, x = 1, x = 2 ....). This is important if the user is concerned that a perfect or near perfect match may be masked by a lower perfect match over a longer range. For example, 7 out of 7 matched sites would not be reported if (1) a set of sequences matched 8 out of 10 sites, and (2) the match criteria allowed 2 mismatches .

  While the invention has been described in detail for purposes of illustration, such details are for that purpose only and depart from the spirit and scope of the invention as defined solely by the appended claims. It will be understood that modifications can be made by those skilled in the art without any problem.

  It is a flowchart which shows each process of a prior art and the polymerase chain reaction (PCR) / ligase detection reaction (LDR) of this invention, and relates to the detection of the germline variation | mutation, such as a point mutation.   FIG. 5 is a flow chart showing the PCR / LDR process of the prior art and the present invention, which relates to the detection of mutations associated with cancer.   The PCR / LDR process of the invention using allele-specific probe addresses for the detection of homozygotes or heterozygotes in two polymorphisms of the same gene (ie allelic differences) FIG.   Schematic diagram illustrating the PCR / LDR process of the present invention using addresses on a common probe for the detection of homozygotes or heterozygotes in two polymorphisms (ie, allelic differences) on the same gene. is there.   FIG. 2 is a schematic diagram illustrating the PCR / LDR process of the present invention using addresses on an allele-specific probe that identifies all possible bases at a given site.   FIG. 4 is a schematic diagram illustrating the PCR / LDR process of the present invention using addresses on a common probe that identifies all bases that can occur at any site.   FIG. 4 is a schematic diagram showing the PCR / LDR process of the present invention using addresses on an allele-specific probe for the purpose of detecting the presence or absence of any base that may be present in two nearby sites.   6 is a schematic diagram illustrating the PCR / LDR process of the present invention using addresses on a common probe for the purpose of detecting the presence or absence of any base that may be present at two nearby sites.   FIG. 6 is a schematic diagram illustrating the PCR / LDR process of the present invention using addresses on an allele-specific probe that discriminate between insertions and deletions.   FIG. 4 is a schematic diagram illustrating the PCR / LDR process of the present invention using addresses on a common probe to identify insertions and deletions.   FIG. 4 is a schematic diagram illustrating the PCR / LDR process of the present invention using allele-specific probe addresses to detect mutations (within a codon) that are few in the majority of normal sequences.   FIG. 4 is a schematic diagram illustrating the PCR / LDR process of the present invention using addresses on a common probe to detect mutations (within a codon) that are few in the majority of normal sequences.   Fig. 4 is a schematic diagram illustrating the PCR / LDR process of the present invention, where the address is located on a common probe and the allelic differences are distinguished by different fluorescent signals F1, F2, F3, and F4.   1 is a schematic diagram showing the PCR / LDR process of the present invention detecting both adjacent alleles and adjacent alleles.   1 is a schematic diagram illustrating the PCR / LDR process of the present invention for detecting all possible single base mutations for a single codon.   p53 chip hybridization and washing conditions are shown.   Figure 3 shows another format for capture of oligonucleotide probes. In FIG. 17B, the portion specific to the localizable array is on an allele-specific probe. Each allele is identified by the capture of a fluorescent signal on addresses Z1 and Z2, respectively. In FIG. 17C, the portion specific to the localizable array is on a common probe and each allele is identified by the capture of fluorescent signals F1 and F2 corresponding to each of the two alleles.   Chemical reactions for covalent modification, grafting, and oligomer attachment to a solid support are shown.   FIG. 3 shows a design according to the invention with 36 tetramers differing by at least 2 bases that can be used to create a series of unique 24mers.   An overview of the PCR / PCR / LDR method for detecting BRCA1 and BRCA2 mutations is shown.   FIG. 6 shows multiplex LDR detection to detect three specific mutations of BRCA1 and BRCA2 in a gel-based assay.   An overview of multiplex LDR detection to detect three specific mutations of BRCA1 and BRCA2 using universal DNA microarrays is presented.   Shown is LDR detection to detect three specific mutations of BRCA1 and BRCA2 on a positionable universal microarray.   FIG. 6 shows p53 chip hybridization indicating the presence of mutations in DNA from colorectal tumors. FIG.   A list of 4633 capture oligonucleotides made in accordance with the present invention is shown.   A list of 465 capture oligonucleotides made in accordance with the present invention is shown.   A list of 96 capture oligonucleotides made in accordance with the present invention is shown.   A list of 65 capture oligonucleotides made in accordance with the present invention is shown.   A list of 4633 capture oligonucleotides (in the form of 20mer PNA) made in accordance with the present invention is shown. Figure 6 shows the distribution of melting temperature (ie Tm) for a list of 96 capture oligonucleotides made according to the present invention. Figure 6 shows the distribution of melting temperature (ie Tm) for a list of 465 capture oligonucleotides made according to the present invention. Figure 5 shows the melting temperature (ie, Tm) distribution for a list of 4633 capture oligonucleotides made in accordance with the present invention. FIG. 6 shows a sorted distribution of melting temperature (ie, Tm) for a list of 4633 capture oligonucleotides made in accordance with the present invention.   Figure 4 shows tetramer usage in a list of 65, 96, 465 and 4633 capture oligonucleotides made in accordance with the present invention.   A computer program is described that compares a target sequence to an array capture probe to ensure that the array capture probe is not designed to hybridize to the target sequence.   Shows LDR detection of 7 specific K-ras mutations in a localizable universal microarray.

Claims (11)

A method for designing multiple capture oligonucleotide probes for use on a support where complementary oligonucleotide probes hybridize with little mismatch, wherein the multiple capture oligonucleotide probes have a narrow range of melting temperatures, comprising: Method including stages:
(1) Each tetramer in the set differs from all other tetramers in the set by at least two nucleotide bases, and (2) no two tetramers complementary to each other are present in the set. (3) tetramers that are palindromic or 2 nucleotide repeats are not present in the set, and (4) tetramers having one or more or three or more nucleotides G or C are not present in the set Providing a first set of tetramers having four nucleotides attached to each other;
Connecting together two to four tetramer groups in the first set to form a collection of multimeric units;
From a set of multimeric units, all multimeric units formed from the same tetramer, and all having a melting temperature less than 4 times the number of tetramers that form the multimeric unit (in Celsius) Removing multimeric units to form a modified set of multimeric units;
Placing a modified set of multimeric units in order of melting temperature and then grouping the multimeric units as a new group of different multimeric units each time the melting temperature is raised twice;
Randomizing the order of groups of multimeric units;
Alternately dividing a group of multimeric units into first and second sub collections based on the order generated by the randomizing step;
Arranging both the first and second subsets in order of increasing or decreasing melting temperature;
Reversing the order of the second subset;
Sequentially coupling the first sub-multimeric unit to the inverted second sub-multimeric unit to form a double multimeric unit set; and from the double multimeric unit set; (1) a double multimer unit having a melting temperature (Celsius) less than 11 times the number of tetramers and a double multimer unit having a melting temperature (Celsius) greater than 15 times the number of tetramers, ( 2) Double multimer unit with 3 identical tetramers connected and 3) Double multimer unit with 4 identical tetramers connected with or without interruption Forming a modified set of.
The method of claim 1 further comprising the following steps:
Placing a modified collection of double multimeric units at a position on the support so that a complementary oligonucleotide to be immobilized on the support can be captured at that position.
The assembly of double multimer units is selected from the double multimer units shown in the following ZIP ID # 1 to 465 , and the multimeric unit of the first sub-assembly according to claim 1, 3. The method according to claim 2, wherein the method is a double multimer unit in the step of binding to an inverted second sub-assembly multimer unit to form an assembly of double multimer units.

The assembly of double multimeric units is selected from the double multimeric units shown in the following ZIP ID # 1-96 , and the first sub-multimeric unit of claim 1 comprises: 3. The method according to claim 2, wherein the method is a double multimer unit in the step of binding to an inverted second sub-assembly multimer unit to form an assembly of double multimer units.

The collection of double multimeric units to be removed has a melting temperature (Celsius) greater than 14 times the number of multimer units and tetramers with a melting temperature (Celsius) less than 12.5 times the number of tetramers 2. The method of claim 1, comprising a multimeric unit.
The method of claim 1, wherein the multimeric unit has 12 mer, the double multimeric unit has 24 mer, and the melting temperature of the double multimeric unit is 75 ° C. to 84 ° C.
The method of claim 1 further comprising the following steps:
Recover double multimer units with a melting temperature (Celsius) less than 11 times the number of tetramers and double multimer units with a melting temperature (Celsius) greater than 15 times the number of tetramers (reclaim) ) Stage;
Separating the recovered double multimeric units, each forming a multimeric unit pair;
Selecting a multimeric unit having a melting temperature (Celsius) greater than 11 times the number of tetramers and less than 17 times the number of tetramers; and reincorporating the selected multimeric unit into the method .
8. The method of claim 7, wherein the steps of collecting, releasing the linkage to form a multimeric unit pair, selecting, and re-installing are repeated.
The first set of tetramers is one selected from the following tetramers and their complements, the first set of tetramers provided in claim 1. Method.

The multimer of the first sub-assembly according to claim 1, wherein the set of double multimer units is selected from the double multimer unit shown in ZIP ID # 1-465 of claim 3 2. The method of claim 1, wherein the unit is a double multimer unit in the step of binding the inverted second sub-assembly multimeric unit to form a double multimeric unit assembly.
A modified set of double multimer units is selected from the double multimer units shown in ZIP ID # 1-96 according to claim 4 and the first sub-unit according to claim 1 2. The method of claim 1, wherein the multimeric unit of the assembly is a double multimeric unit in the step of binding the multimeric unit of the second sub-assembly to the inverted second multimeric unit to form an assembly of double multimeric units.

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